Long lasting drug formulations

ABSTRACT

The present invention is directed to long-lasting therapeutic formulations and their methods of use wherein the formulation comprises a genetically modified micro-organ that comprises a vector which comprises a nucleic acid sequence operably linked to one or more regulatory sequences, wherein the nucleic acid sequence encodes a therapeutic polypeptide, such as erythropoietin or interferon alpha.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/898,481, filed Sep. 12, 2007, which claims the benefit of U.S.Patent Application Ser. No. 60/844,351, filed on Sep. 14, 2006, whichare incorporated in their entirety herein by reference.

BACKGROUND OF THE INVENTION

Therapeutic agents can be delivered orally, transdermally, byinhalation, by injection or by depot with slow release. However, themethod of delivery is limited by the processing that the agent issubjected to in the recipient, by the requirement for frequentadministration, and limitations on the size of molecules that can beutilized. For some of the methods, the amount of therapeutic agentvaries between administrations.

Protein production techniques which involve the sub-cloning of a desirednucleic acid sequence/fragment into a vector which is subsequently usedfor modifying specific host cells, which are meant to produce thedesired protein for further purification steps are limited in the amountof protein expressed, protein secretion, post-translationalmodifications (such as glycosylation and the accurate folding of theprotein), etc. Moreover, even if a high-level of protein productioncould be achieved, large quantities of the recombinant protein must thenbe produced and purified to be free of contaminants. Development of apurification scheme is a very lengthy process. And once purifiedrecombinant protein has been obtained, it must be further formulated torender it stable and acceptable for introduction into animals or humans.Furthermore, even formulated, purified recombinant proteins have afinite shelf life due to maintenance and storage limitations; oftenrequiring repeated purification and formulation of more protein. Theprocess of developing an appropriate formulation is time consuming,difficult, and costly, as well.

Thus, there is a widely recognized need for long-lasting protein-basedtherapeutic molecules that have the requisite post-translationalmodifications to preserve their biological activity, which are producedinexpensively and quickly without the need for the laborious and costlymethods typically associated with obtaining high-levels of recombinantproteins.

Some researchers have attempted to obtain in vivo expression ofrecombinant gene products via gene therapy. Typically viral vectors areused to transduce cells in vivo to express recombinant gene products.These viral-based vectors have advantageous characteristics, such as thenatural ability to infect the target tissue. However, retrovirus-basedvectors require integration within the genome of the target tissue toallow for recombinant product expression (with the potential to activateresident oncogenes) and can only be used to transduce actively dividingtissues. Viral vectors are also often no able to sustain long-termtransgene expression, which may be due at least in part to theirelimination due to secondary host immune responses.

Accordingly, there remains a need in the art for recombinant geneproduct formulations that have consistently high expression levelslasting for several weeks or more and for methods of using thoseformulations to treat disease.

SUMMARY OF THE INVENTION

The invention provides, in one embodiment, a long-lasting therapeuticformulation comprising a genetically modified micro-organ, saidmicro-organ comprising a vector comprising a nucleic acid sequenceoperably linked to one or more regulatory sequences, wherein saidnucleic acid sequence encodes a therapeutic polypeptide and whereby saidformulation increases expression levels of said therapeutic polypeptideby more than 5% over basal level and said increase is maintained forgreater than one month. In one embodiment, the vector is ahelper-dependent adenovirus vector. In one embodiment, the therapeuticpolypeptide is erythropoietin, while in another embodiment, thetherapeutic polypeptide is interferon alpha, which in one embodiment, isinterferon alpha 2b.

In another embodiment, the invention provides a method of providing atherapeutic polypeptide to a subject in need over a sustained periodcomprising providing one or more genetically modified micro-organs, saidmicro-organs comprising a vector comprising a nucleic acid sequenceoperably linked to one or more regulatory sequences; and implanting saidgenetically modified micro-organ in said subject, wherein said nucleicacid sequence encodes a therapeutic polypeptide and whereby saidformulation increases expression levels of said therapeutic polypeptideby more than 5% over basal level and said increase is maintained forgreater than one month. In one embodiment, the vector is ahelper-dependent adenovirus vector.

In one embodiment, the therapeutic polypeptide is erythropoietin, whilein another embodiment, the therapeutic polypeptide is interferon alpha,which in one embodiment, is interferon alpha 2b.

In another embodiment, the subject in need is suffering from anemia. Inanother embodiment, the subject in need is suffering from an infection.In another embodiment, the subject in need is suffering from cancer.

In another embodiment, the invention provides a nucleic acid sequencewith greater than 85% homology to SEQ ID No: 1, a vector comprising sucha nucleic acid sequence, and a cell comprising such as vector.

In another embodiment, the invention provides a nucleic acid sequencewith greater than 85% homology to SEQ ID No: 2, a vector comprising sucha nucleic acid sequence, and a cell comprising such as vector.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents levels of recombinant optimized human interferon-alpha(IFNα) produced in vitro by the formulations of the instant invention.

FIG. 2 presents levels of recombinant human erythropoietin (hEPO)produced in vitro by the formulations of the instant invention.HD-Ad-CAG-wt-hEPO GMMO titration (A). Micro-organs were transduced withincreasing dilutions of HD-Ad-CAG-wt-hEPO virus: 1:25; 1:100; and 1:1000dilutions. Ad5/CMV/wt-hEPO was diluted to a working concentration of1:10 and 1:50. A comparison between GMMOs produced from two differentskins, H-1 and H-2 (B). Micro-organs were transduced withHD-Ad-CAG-wt-hEPO 1:25. Bars indicate the hEPO concentration measured byELISA in the culture media that was collected and replaced every 3-4days.

FIG. 3 presents the percent of peak erythropoietin (EPO) expressionlevels in vitro from optimized formulations comprising EPO-expressinggutless adenovirus and micro-organs comprising EPO-expressingadenovirus-5. Micor-organs were transduced with HD-Ad-CAG-hEPO at 1:25or with Ad5/CMV/hEPO at 1:10.

FIG. 4 presents erythropoietin (EPO) expression levels in vitro fromformulations comprising optimized and non-optimized EPO-expressinggutless adenovirus. Micro-organs were transduced with a working dilutionof 1:100 viral particles. Bars indicate the hEPO concentration measuredby ELISA in the culture media that was collected and replaced every 3-4days.

FIG. 5 presents erythropoietin (EPO) expression levels in vitro fromformulations comprising EPO-expressing gutless adenovirus downstream ofa CAG or CMV promoter.

FIG. 6 presents levels of recombinant human erythropoietin produced invivo in SCID mice (A) and in vitro (B) by the formulations of theinstant invention in vitro and the associated changes in hematocrit (A).Ten mice/group were implanted subcutaneously with GMMOs. The hEPO levels(mU/ml) and the corresponding % hematocrit that were measured in theserum of mice that were implanted with GMMOs transduced withadenovirus-hEPO, helper-dependent adenovirus-hEPO, and helper-dependentadenovirus-optimized hEPO and with non-transduced GMMOs are presented.Bleeds were done every 10 days (A). Hematocrit was measured by thecentrifugation method and serum hEPO levels in the blood were measuredby a hEPO ELISA kit. Non-implanted GMMOs were maintained in culture andlevels of EPO were measured (B)

DETAILED DESCRIPTION OF THE PRESENT INVENTION

In some embodiments, the instant invention is directed to long-lastingtherapeutic formulations comprising a genetically modified, tissue-basedmicro-organ comprising a vector comprising a nucleic acid sequenceencoding a therapeutic polypeptide, such as erythropoietin or interferonalpha, operably linked to one or more regulatory sequences and theirmethods of use.

The invention provides, in one embodiment, a long-lasting therapeuticformulation comprising a genetically modified micro-organ, saidmicro-organ comprising a vector comprising a nucleic acid sequenceoperably linked to one or more regulatory sequences, wherein saidnucleic acid sequence encodes a therapeutic polypeptide and whereby theexpression level of the therapeutic polypeptide is increased by morethan 5% over basal level and said increase is maintained for greaterthan one month. In another embodiment, the expression level of thetherapeutic polypeptide is increased by more than 5% over basal leveland said increase is maintained for greater than six months.

In another embodiment, this invention provides a long-lastingtherapeutic formulation comprising a genetically modified micro-organ,said micro-organ comprising a vector comprising a nucleic acid sequenceoperably linked to one or more regulatory sequences, wherein saidnucleic acid sequence encodes a therapeutic polypeptide and whereby theexpression level of the therapeutic polypeptide is increased by morethan 5% over basal level and said increase is maintained for greaterthan one month and wherein said vector is a helper-dependent adenovirusvector.

In another embodiment, the invention provides a long-lasting therapeuticformulation comprising a genetically modified micro-organ, saidmicro-organ comprising a vector comprising a nucleic acid sequenceoperably linked to one or more regulatory sequences, wherein saidnucleic acid sequence encodes a therapeutic polypeptide and whereby theexpression level of the therapeutic polypeptide is increased by morethan 5% over basal level and said increase is maintained for greaterthan one month in an immuno-competent host.

In another embodiment, the invention provides a long-lasting therapeuticformulation comprising a genetically modified micro-organ, saidmicro-organ comprising a vector comprising a nucleic acid sequenceoperably linked to one or more regulatory sequences, whereby theexpression level of the therapeutic nucleic acid is increased by morethan 5% over basal levels. In one embodiment, the expression level ofthe therapeutic nucleic acid is increased by more than 5% over basallevels in an immuno-competent host, while in another embodiment, saidvector is a helper-dependent adenovirus vector.

In one embodiment, the invention provides a long-lasting therapeuticformulation and methods of use thereof where the formulation comprises agenetically modified micro-organ. In one embodiment, the term“micro-organ” as used herein, refers in one embodiment, to an isolatedtissue or organ structure derived from or identical to an explant thathas been prepared in a manner conducive to cell viability and function.In one embodiment, a micro-organ maintains at least some in vivostructures, or in another embodiment, interactions, similar to thetissues or organ from which it is obtained, In another embodiment,micro-organs retain the micro-architecture and the three dimensionalstructure of the tissue or organ from which they were derived and havedimensions selected so as to allow passive diffusion of adequatenutrients and gases to cells within the micro-organ and diffusion ofcellular waste out of the cells of the micro-organ so as to minimizecellular toxicity and concomitant cell death due to insufficientnutrition and/or accumulation of waste. In one embodiment, a micro-organis a sliver of dermal tissue.

In one embodiment, a micro-organ is 1-2 mm in diameter and 30-40 mm inlength. In another embodiment, the diameter of a micro-organ may be, forexample, 1-3 mm, 1-4 mm, 2-4 mm, 0.5-3.5 mm, or 1.5-10 mm. In anotherembodiment the diameter of a micro-organ may be, for example,approximately 2 mm or approximately 1.5 mm. In another embodiment, thelength of the micro-organ may be 5-100 mm, 10-60 mm, 20-60 mm, 20-50 mm,20-40 mm, 20-100 mm, 30-100 mm, 40-100 mm, 50-100 mm, 60-100 mm, 70-100mm, 80-100 mm, or 90-100 mm. In another embodiment, the length of themicro-organ may be approximately 20 mm, approximately 30 mm,approximately 40 mm, or approximately 50 mm. In one embodiment, amicro-organ is smaller than 1.5 cm², and in another embodiment, lessthan 1 cm². In another embodiment, the diameter is less than 1.5 cm, andin another embodiment, the length is less than 1.5 cm.

In one embodiment, a micro-organ is an explant. In one embodiment, amicro-organ is tissue-derived. In another embodiment, a micro-organ is asection or portion or part of a tissue. In another embodiment, amicro-organ is a section or portion or part of an organ. A micro-organcan be distinguished from a skin graft, in one embodiment, in that it isspecifically designed to survive for long periods of time in vivo and invitro and, in another embodiment, in that its dimensions arespecifically selected so as to allow passive diffusion of adequatenutrients and gases to cells within the micro-organ and diffusion ofcellular waste out of the cells of the micro-organ, which in oneembodiment minimizes cellular toxicity and concomitant cell death due toinsufficient nutrition and/or accumulation of waste. Thus, in oneembodiment, a micro-organ is not a skin graft. In another embodiment, amicro-organ can be distinguished from a collection of isolated cells,which in one embodiment, are grown on a natural or artificial scaffold,in that micro-organs maintain the micro-architecture and the threedimensional structure of the tissue or organ from which they werederived. Thus, in one embodiment, a micro-organ is not one or more celltypes grown on a scaffold.

A detailed description of micro-organs can be found in US-2003-0152562,which is incorporated herein by reference in its entirety.

Earlier patents (WO 03/006669, WO 03/03585, WO 04/0993631, which areincorporated herein by reference) described micro-organs, which can bemodified to express a gene product of interest, that may be sustainedoutside the body in an autonomously functional state for an extendedperiod of time, and may then be implanted subcutaneously or in otherlocations within the body for the purpose of treating diseases ordisorders. However, the micro-organs of the present inventionunexpectedly showed a much longer-term expression profile of a geneproduct of interest in vitro and in vivo.

As used herein, the term “explant” refers, in one embodiment, to atissue or organ or a portion thereof removed from its natural growthsite in an organism and placed in a culture medium for a period of time.In one embodiment, the tissue or organ is viable, in another embodiment,metabolically active, or a combination thereof.

As used herein, the term “microarchitecture” refers, in one embodiment,to a characteristic of the explant in which some or all of the cells ofthe tissue explant maintain, in vitro, physical and/or functionalcontact with at least one cell or non-cellular substance with which theywere in physical and/or functional contact in vivo.

In another embodiment, micro-organ explants maintain thethree-dimensional structure of the tissue or organ from which they werederived. In one embodiment, micro-organ explants retain the spatialinteractions, e.g. cell-cell, cell-matrix and cell-stromal interactions,and the orientation of the tissue from which they were derived. In oneembodiment, preservation of spatial interactions such as described abovepermit the maintenance of biological functions of the explant, such assecretion of autocrine and paracrine factors and other extracellularstimuli, which in one embodiment, provide long term viability to theexplant. In one embodiment, at least some of the cells of themicro-organ explant maintain, in vitro, their physical and/or functionalcontact with at least one cell or non-cellular substance with which theywere in physical and/or functional contact in vivo. In one embodiment,some of the cells refers to at least about 50%, in another embodiment,at least about 60%, in another embodiment at least about 70%, in anotherembodiment, at least about 80%, and in another embodiment, at leastabout 90% or more of the cells of the population. In another embodiment,the cells of the explant maintain at least one biological activity ofthe organ or tissue from which they are isolated.

In some embodiments, any of the formulation of this invention willcomprise a genetically modified micro-organ, in any form or embodimentas described herein. In some embodiments, any of the formulations ofthis invention will consist of a genetically modified micro-organ, inany form or embodiment as described herein. In some embodiments, of thecompositions of this invention will consist essentially of a geneticallymodified micro-organ, in any form or embodiment as described herein. Insome embodiments, the term “comprise” refers to the inclusion of theindicated active agent, such as the genetically modified micro-organ, aswell as inclusion of other active agents, and pharmaceuticallyacceptable carriers, excipients, emollients, stabilizers, etc., as areknown in the pharmaceutical industry. In some embodiments, the term“consisting essentially of” refers to a composition, whose only activeingredient is the indicated active ingredient, however, other compoundsmay be included which are for stabilizing, preserving, etc. theformulation, but are not involved directly in the therapeutic effect ofthe indicated active ingredient. In some embodiments, the term“consisting essentially of” may refer to components which facilitate therelease of the active ingredient. In some embodiments, the term“consisting” refers to a composition, which contains the activeingredient and a pharmaceutically acceptable carrier or excipient.

Similarly, in some embodiments, the vector of and for use in the methodsof the present invention comprise a nucleic acid sequence operablylinked to one or more regulatory sequences, wherein said nucleic acidsequence encodes a therapeutic polypeptide. In another embodiment, thevector consists essentially of such a nucleic acid sequence, and inanother embodiment, the vector consists of such a nucleic acid sequence.

Examples of mammals from which the micro-organs can be isolated includehumans and other primates, swine, such as wholly or partially inbredswine (e.g., miniature swine, and transgenic swine), rodents, etc.Micro-organs may be processed from tissue from a variety of organs,which in one embodiment is the skin, the dermis, the lymph system, thepancreas, the liver, the gallbladder, the kidney, the digestive tract,the respiratory tract, the reproductive system, the urinary tract, theblood, the bladder, the cornea, the prostate, the bone marrow, thethymus, the spleen, or a combination thereof. Explants from these organsmay comprise islet of Langerhan cells, hair follicles, glands,epithelial and connective tissue cells, or a combination thereofarranged in a microarchitecture similar to the microarchitecture of theorgan from which the explant was obtained. In one embodiment, themicroarchitecture of the organ from which the explant was obtained maybe discerned or identified in the micro-organ explant using materials,apparati, and/or methods known in the art.

In one embodiment, the present invention provides a formulation andmethods of use thereof comprising a genetically modified micro-organ. Inone embodiment, the term “genetically modified micro-organ” or “GMMO”refers to a micro-organ that expresses at least one recombinant geneproduct. In other embodiments, reference to a micro-organ does notnecessarily refer to a non-genetically modified micro-organ, but mayalso refer in some instances to a genetically modified micro-organ aswill be clear from the context to one of skill in the art. In oneembodiment, the phrase “gene product” refers to proteins, polypeptides,peptides and functional RNA molecules. In one embodiment, the geneproduct encoded by the nucleic acid molecule is the desired gene productto be supplied to a subject. Examples of such gene products includeproteins, peptides, glycoproteins and lipoproteins normally produced bycells of the recipient subject. In one embodiment, the gene product isnot naturally occurring in the organism from which the micro-organ washarvested and/or in the organism in which the GMMO is implanted, whilein another embodiment, the gene product is naturally occurring. In oneembodiment, the gene product of the GMMO is similar or identical to agene product endogenously expressed by one or more cells of themicro-organ. In one embodiment, genetic modification increases the levelof a gene product that would be produced in a non-genetically modifiedmicro-organ. In another embodiment, the gene product expressed by theGMMO is not similar or identical to a gene product endogenouslyexpressed by one or more cells of the micro-organ. In anotherembodiment, the gene product encoded by the nucleic acid moleculeencodes a molecule that directly or indirectly controls expression of agene of interest. In another embodiment, the gene product encoded by thenucleic acid molecule upregulates or downregulates the expression levelsof the desired gene product to be supplied to a subject.

In another embodiment, genetic modification of a micro-organ may modifythe expression profile of an endogenous gene. This may be achieved, forexample, by introducing an enhancer, or a repressible or inducibleregulatory element for controlling the expression of an endogenous gene.

Any methodology known in the art can be used for genetically alteringthe micro-organ explant. Any one of a number of different vectors can beused, such as viral vectors, plasmid vectors, linear DNA, etc., as knownin the art, to introduce an exogenous nucleic acid fragment encoding atherapeutic agent into target cells and/or tissue. These vectors can beinserted, for example, using infection, transduction, transfection,calcium-phosphate mediated transfection, DEAE-dextran mediatedtransfection, electroporation, liposome-mediated transfection, biolisticgene delivery, liposomal gene delivery using fusogenic and anionicliposomes (which are an alternative to the use of cationic liposomes),direct injection, receptor-mediated uptake, magnetoporation, ultrasound,or any combination thereof, as well as other techniques known in the art(for further detail see, for example, “Methods in Enzymology” Vol.1-317, Academic Press, Current Protocols in Molecular Biology, AusubelF. M. et al. (eds.) Greene Publishing Associates, (1989) and inMolecular Cloning: A Laboratory Manual, 2nd Edition, Sambrook et al.Cold Spring Harbor Laboratory Press, (1989), or other standardlaboratory manuals). The polynucleotide segments encoding sequences ofinterest can be ligated into an expression vector system suitable fortransducing mammalian cells and for directing the expression ofrecombinant products within the transduced cells. The introduction ofthe exogenous nucleic acid fragment is accomplished by introducing thevector into the vicinity of the micro-organ. Once the exogenous nucleicacid fragment has been incorporated into the cells using any of thetechniques described above or known in the art, the production and/orthe secretion rate of the therapeutic agent encoded by the nucleic acidfragment can be quantified. In one embodiment, the term “exogenous”refers to a substance that originated outside, for example a nucleicacid that originated outside of a cell or tissue.

In one embodiment, a micro-organ of the formulation and methods of thepresent invention comprises a vector, which in one embodiment,facilitates recombinant gene expression. In one embodiment, the vectoris a non-immunogenic gene transfer agent such as a nonviral vector (e.g.DNA plasmids or minicircle DNA), a “gutless” viral vector i.e. withoutendogenous genes (which in one embodiment, is due to a deletion, whilein another embodiment, due to an insertion, substitution or deletion ina gene that prevents gene expression), a helper-dependent adenovirus(HDAd) vector, or adeno associated virus AAV (which in one embodiment issingle stranded and in another embodiment, double stranded). In anotherembodiment, said formulation is so chosen such that recombinant geneexpression results in lack of toxicity or immune-mediated rejection ofthe gene product by the micro-organ. In one embodiment, the vector isvirally derived, and in another embodiment, the vector is a plamid. Inone embodiment, the virally-derived vector is derived from adenovirus,which in one embodiment, is helper-dependent adenovirus, while inanother embodiment, the virally-derived vector is derived fromadenovirus-associated vector, as is described hereinbelow.

In one embodiment, the term “vector” or “expression vector” refers to acarrier molecule into which a nucleic acid sequence can be inserted forintroduction into a cell where it can be replicated. In one embodiment,the nucleic acid molecules are transcribed into RNA, which in some casesare then translated into a protein, polypeptide, or peptide. In othercases, RNA sequences are not translated, for example, in the productionof antisense molecules or ribozymes. In one embodiment, expressionvectors can contain a variety of “control sequences” which refer tonucleic acid sequences necessary for the transcription and possiblytranslation of an operably linked coding sequence in a particular hostcell. In another embodiment, a vector further includes an origin ofreplication. In one embodiment the vector may be a shuttle vector, whichin one embodiment can propagate both in prokaryotic and eukaryoticcells, or in another embodiment, the vector may be constructed tofacilitate its integration within the genome of an organism of choice.The vector, in other embodiments may be, for example, a plasmid, abacmid, a phagemid, a cosmid, a phage, a virus or an artificialchromosome. In one embodiment, the vector is a viral vector, which inone embodiment may be a bacteriophage, mammalian virus, or plant virus.

In one embodiment, the viral vector is an adenoviral vector. In anotherembodiment, the adenovirus may be of any known serotype or subgroup.

In one embodiment, some advantages of using an adenoviral vector as agene transfer vector are: its mid-sized genome, ease of manipulation,high titer, wide target-cell range and high infectivity. Both ends ofthe adenoviral genome contain 100-200 base pair inverted repeats (ITRs),which are cis elements necessary for viral DNA replication andpackaging. The early (E) and late (L) regions of the genome containdifferent transcription units that are divided by the onset of viral DNAreplication. The E1 region (E1A and E1B) encodes proteins responsiblefor the regulation of transcription of the viral genome and a fewcellular genes. The expression of the E2 region (E2A and E2B) results inthe synthesis of the proteins for viral DNA replication. These proteinsare involved in DNA replication, late gene expression and host cellshut-off. The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP, (located at 16.8 m.u.) is particularly efficient during thelate phase of infection, and all the mRNAs issued from this promoterpossess a 5′-tripartite leader (TPL) sequence which makes them preferredmRNAs for translation.

In another embodiment, the adenoviral vector is a helper-dependentadenoviral vector, which in another embodiment, is synonymous withgutless, gutted, mini, fully deleted, high-capacity, Δ, or pseudoadenovirus, and which in another embodiment are deleted of all viralcoding sequences except for sequences supporting DNA replication, whichin one embodiment, comprise the adenovirus inverted terminal repeats andpackaging sequence (ψ). In another embodiment, helper-dependentadenoviruses express no viral proteins. In one embodiment, ahelper-dependent adenoviral vector comprises only the cis-actingelements of the adenovirus required to replicate and package the vectorDNA. In one embodiment, a helper-dependent adenoviral vector comprisesapproximately 500 bp of wild-type adenovirus sequence. In anotherembodiment, the adenoviral vector additionally comprises stuffer DNA tomeet the minimum requirement for a genome size of 27.7 kb, which in oneembodiment is required for efficient packaging into the adenoviruscapsid. In one embodiment, non-coding mammalian DNA, with minimal repeatsequences, is used as stuffer DNA. In another embodiment, stuffer DNAcomprises non-mammalian DNA, which in one embodiment, is HPRT and/orC346 cosmid sequences.

In one embodiment, helper-dependent adenoviruses display high-efficiencyin vivo transduction, high-level transgene expression, are able tomaintain long-term transgene expression, in one embodiment, by avoidingchronic toxicity due to residual expression of viral proteins, or acombination thereof. In another embodiment, helper-dependentadenoviruses have high titer production, efficient infection of a broadrange of cell types, the ability to infect dividing and nondividingcells, or a combination thereof. In another embodiment, ahelper-dependent adenovirus for use in the methods of the instantinvention does not induce a strong adaptive immune response to animplanted micro-organ, which in one embodiment, is characterized by thegeneration of adeno-specific MHC class I restricted CD8 cytotoxic Tlymphocytes (CTL) in immunocompetent hosts, which in one embodiment,would limit the duration of transgene expression and in anotherembodiment, would result in adenovirus vector clearance within severalweeks. In another embodiment, a helper-dependent adenovirus for use inthe methods of the instant invention does not induce high cytotoxic Tcell levels (as may be measured in one embodiment by positive CD8staining, as is known in the art), and, in another embodiment, does notinduce high helper T cell levels (as may be measured in one embodimentby positive CD4 stain, as is known in the art).

In another embodiment, helper-dependent adenoviruses have a lower riskof germ line transmission and insertional mutagenesis that may causeoncogenic transformation, because the vector genome does not integrateinto the host cell chromosomes. In one embodiment, the cloning capacityof helper-dependent adenoviruses is very large (in one embodiment,approximately 37 kb, in another embodiment, approximately 36 kb),allowing for the delivery of whole genomic loci, multiple transgenes,and large cis-acting elements to enhance, prolong, and regulatetransgene expression.

In one embodiment, the helper-dependent adenovirus system for use withthe compositions and in the methods of the present invention is similarto that described in Palmer and Ng, 2003 (Mol Ther 8:846) and in Palmerand Ng, 2004 (Mol Ther 10:792), which are hereby incorporated herein byreference in their entirety. In one embodiment, there is a stalersequence inserted into the E3 region of the helper virus component ofthe helper-dependent adenovirus system to minimize recombination betweenthe helper adenovirus and the helper-dependent adenovirus to producereplication competent adenovirus.

In one embodiment, formulations of the instant invention comprisinghelper-dependent adenoviral vectors demonstrate long-term, high in vitro(FIGS. 1, 2, and 6B) and in vivo (FIG. 6A) expression levels of EPO andIFN-alpha. In another embodiment, formulations of the instant inventioncomprising helper-dependent adenoviral vectors demonstrate an increasedpercent of peak EPO expression levels for at least 100 dayspost-transduction compared to micro-organs comprising adenovirus-5 (FIG.3). Without being bound by theory, one factor that may contribute to thelong-lasting, high levels of gene product from micro-organs of theinstant invention is use of a helper-dependent adenovirus vector, whichis non-toxic to tissue and non-immunogenic within the formulations ofthe present invention.

In another embodiment, the adenoviral vector is E1-deleted, while inanother embodiment, the adenoviral vector additionally comprisesdeletions for E2, E3, E4, or a combination thereof.

In another embodiment, the viral vector is an adeno-associated viralvector (AAV). In one embodiment, AAV is a parvovirus, discovered as acontamination of adenoviral stocks. It is a ubiquitous virus (antibodiesare present in 85% of the US human population) that has not been linkedto any disease. It is also classified as a dependovirus, because itsreplication is dependent on the presence of a helper virus, such asadenovirus. At least nine serotypes have been isolated, of which AAV-2is the best characterized. AAV has a single-stranded linear DNA that isencapsidated into capsid proteins VP1, VP2 and VP3 to form anicosahedral virion of 20 to 24 nm in diameter.

In one embodiment, the AAV DNA is approximately 4.7 kilobases long. Inone embodiment, it contains two open reading frames and is flanked bytwo ITRs. There are two major genes in the AAV genome: rep and cap. Therep gene codes for proteins responsible for viral replications, whereascap codes for capsid protein VP1-3. Each ITR forms a T-shaped hairpinstructure. These terminal repeats are the only essential cis componentsof the AAV for chromosomal integration. Therefore, in one embodiment,the AAV can be used as a vector with all viral coding sequences removedand replaced by the cassette of genes for delivery.

In one embodiment, when using recombinant AAV (rAAV) as an expressionvector, the vector comprises the 145-bp ITRs, which are only 6% of theAAV genome, which in one embodiment, leaves space in the vector toassemble a 4.5-kb DNA insertion.

In one embodiment, AAV is safe in that it is not considered pathogenicnor is it associated with any disease. The removal of viral codingsequences minimizes immune reactions to viral gene expression, andtherefore, rAAV evokes only a minimal inflammatory response, if any. Inanother embodiment, AAV vector is double-stranded, while in anotherembodiment, AAV vector is self-complementary, which in one embodiment,bypasses the requirement of viral second-strand DNA synthesis, which inone embodiment, results in early transgene expression.

In another embodiment, the viral vector is a retroviral vector. Theretroviruses are a group of single-stranded RNA viruses characterized byan ability to convert their RNA to double-stranded DNA in infected cellsby a process of reverse-transcription. The resulting DNA then stablyintegrates into cellular chromosomes as a provirus and directs synthesisof viral proteins. The integration results in the retention of the viralgene sequences in the recipient cell and its descendants. The retroviralgenome contains three genes, gag, pol, and env that code for capsidproteins, polymerase enzyme, and envelope components, respectively. Asequence found upstream from the gag gene contains a signal forpackaging of the genome into virions. Two long terminal repeat (LTR)sequences are present at the 5′ and 3′ ends of the viral genome. Thesecontain strong promoter and enhancer sequences and are also required forintegration in the host cell genome.

In order to construct a retroviral vector in one embodiment, a nucleicacid encoding one or more oligonucleotide or polynucleotide sequences ofinterest is inserted into the viral genome in the place of certain viralsequences to produce a virus that is replication-defective. In order toproduce virions, a packaging cell line containing the gag, pol, and envgenes but without the LTR and packaging components is constructed. Whena recombinant plasmid containing a cDNA, together with the retroviralLTR and packaging sequences is introduced into this cell line (bycalcium phosphate precipitation, for example), the packaging sequenceallows the RNA transcript of the recombinant plasmid to be packaged intoviral particles, which are then secreted into the culture media. Themedia containing the recombinant retroviruses is then collected,optionally concentrated, and used for gene transfer. Retroviral vectorsare able to infect a broad variety of cell types. However, integrationand stable expression require the division of host cells.

In other embodiments, the viral vector is derived from a virus such asvaccinia virus, lentivirus, polio virus, hepatitis virus, papillomavirus, cytomegalovirus, simian virus, or herpes simplex virus.

In certain embodiments of the invention, the vector comprising a nucleicacid sequence may comprise naked recombinant DNA or plasmids. Transferof the construct may be performed by any method which physically orchemically permeabilizes the cell membrane. In one embodiment, thevector is a mini-circle DNA, which in one embodiment, is a supercoiledDNA molecule for non-viral gene transfer, which has neither a bacterialorigin of replication nor an antibiotic resistance marker. In anotherembodiment, mini-circle DNA comprises no bacterial control regions fromgene delivery vectors during the process of plasmid production. They arethus smaller and potentially safer than other plasmids used in genetherapy. In one embodiment, mini-circle DNA produce high yield, aresimple to purify, and provide robust and persistent transgeneexpression.

Construction of vectors using standard recombinant techniques is wellknown in the art (see, for example, Maniatis, et al., Molecular Cloning,A Laboratory Manual (Cold Spring Harbor, 1990) and Ausubel, et al.,1994, Current Protocols in Molecular Biology (John Wiley & Sons, 1996),both incorporated herein by reference).

In another embodiment, a vector further comprises an insertion of aheterologous nucleic acid sequence encoding a marker polypeptide. Themarker polypeptide may comprise, for example, yECitrine, greenfluorescent protein (GFP), DS-Red (red fluorescent protein), secretedalkaline phosphatase (SEAP), β-galactosidase, chloramphenicol acetyltransferase, luciferase, GFP/EGFP, human growth hormone, or any numberof other reporter proteins known to one skilled in the art.

In another embodiment, the vectors may comprise one or more genes ofinterest. Thus, in one embodiment, a vector of the instant invention maycomprise a gene of interest, which in one embodiment, is erythropoietinor interferon alpha2b, which in one embodiment, expresses a marker, andin another embodiment, is linked in frame to a marker, which in oneembodiment allows identification of the gene product of interest and inanother embodiment, allows the distinction between a gene product ofinterest produced by a micro-organ and a similar gene product producedendogenously by host cells outside of the micro-organ(s).

In one embodiment, a vector comprising a nucleic acid encoding atherapeutic polypeptide of the instant invention is introduced into amicro-organ. There are a number of techniques known in the art forintroducing cassettes and/or vectors into cells, for affecting themethods of the present invention, such as, but not limited to: directDNA uptake techniques, and virus, plasmid, linear DNA or liposomemediated transduction, receptor-mediated uptake and magnetoporationmethods employing calcium-phosphate mediated and DEAE-dextran mediatedmethods of introduction, electroporation or liposome-mediatedtransfection, (for further detail see, for example, “Methods inEnzymology” Vol. 1-317, Academic Press, Current Protocols in MolecularBiology, Ausubel F. M. et al. (eds.) Greene Publishing Associates,(1989) and in Molecular Cloning: A Laboratory Manual, 2nd Edition,Sambrook et al. Cold Spring Harbor Laboratory Press, (1989), or otherstandard laboratory manuals).

In one embodiment, bombardment with nucleic acid coated particles may bea method for transferring a naked DNA expression construct into cells.This method depends on the ability to accelerate DNA-coatedmicro-projectiles to a high velocity allowing them to pierce cellmembranes and enter cells without killing them. Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force. The micro-projectiles used havecomprised biologically inert or biocompatible substances such astungsten or gold beads. It is to be understood that any of these methodsmay be utilized for introduction of the desired sequences into cells,and cells thereby produced are to be considered as part of thisinvention, as is their use for effecting the methods of this invention.

In one embodiment, the vectors of the formulations and methods of theinstant invention comprise a nucleic acid sequence. As used herein, theterm “nucleic acid” refers to polynucleotide or to oligonucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA) or mimetic thereof. The term should also be understood to include,as equivalents, analogs of RNA or DNA made from nucleotide analogs, and,as applicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotide. This term includesoligonucleotides composed of naturally occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

In one embodiment, the term “nucleic acid” or “oligonucleotide” refersto a molecule, which may include, but is not limited to, prokaryoticsequences, eukaryotic mRNA, cDNA from eukaryotic mRNA, genomic DNAsequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNAsequences. The term also refers to sequences that include any of theknown base analogs of DNA and RNA.

The nucleic acids can be produced by any synthetic or recombinantprocess, which are well known in the art. Nucleic acids can further bemodified to alter biophysical or biological properties by means oftechniques known in the art. For example, the nucleic acid can bemodified to increase its stability against nucleases (e.g.,“end-capping”), or to modify its solubility, or binding affinity tocomplementary sequences. These nucleic acids may comprise the vector,the expression cassette, the promoter sequence, the gene of interest, orany combination thereof. In another embodiment, its lipophilicity may bemodified, which, in turn, will reflect changes in the systems employedfor its delivery, and in one embodiment, may further be influenced bywhether such sequences are desired for retention within, or permeationthrough the skin, or any of its layers. Such considerations mayinfluence any compound used in this invention, in the methods andsystems described.

In one embodiment, DNA can be synthesized chemically from the fournucleotides in whole or in part by methods known in the art. Suchmethods include those described in Caruthers (1985; Science230:281-285). DNA can also be synthesized by preparing overlappingdouble-stranded oligonucleotides, filling in the gaps, and ligating theends together (see, generally, Sambrook et al. (1989; MolecularCloning—A Laboratory Manual, 2nd Edition. Cold Spring Habour LaboratoryPress, New York)). In another embodiment, inactivating mutations may beprepared from wild-type DNA by site-directed mutagenesis (see, forexample, Zoller et al. (1982; DNA. 1984 December; 3(6):479-88); Zoller(1983); and Zoller (1984; DNA. 1984 December; 3 (6):479-88); McPherson(1991; Directed Mutagenesis: A Practical Approach. Oxford UniversityPress, NY)). The DNA obtained can be amplified by methods known in theart. One suitable method is the polymerase chain reaction (PCR) methoddescribed in Saiki et al. (1988; Science. 1988 Jan. 29;239(4839):487-491), Mullis et al., U.S. Pat. No. 4,683,195, and Sambrooket al. (1989).

Methods for modifying nucleic acids to achieve specific purposes aredisclosed in the art, for example, in Sambrook et al. (1989). Moreover,the nucleic acid sequences of the invention can include one or moreportions of nucleotide sequence that are non-coding for the protein ofinterest. Variations in DNA sequences, which are caused by pointmutations or by induced modifications (including insertion, deletion,and substitution) to enhance the activity, half-life or production ofthe polypeptides encoded thereby, are also encompassed in the invention.

The formulations of this invention may comprise nucleic acids, in oneembodiment, or in another embodiment, the methods of this invention mayinclude delivery of the same, wherein, in another embodiment, thenucleic acid is a part of a vector.

The efficacy of a particular expression vector system and method ofintroducing nucleic acid into a cell can be assessed by standardapproaches routinely used in the art as described hereinbelow.

As will be appreciated by one skilled in the art, a fragment orderivative of a nucleic acid sequence or gene that encodes for a proteinor peptide can still function in the same manner as the entire wild typegene or sequence Likewise, forms of nucleic acid sequences can havevariations as compared to wild type sequences, nevertheless encoding theprotein or peptide of interest, or fragments thereof, retaining wildtype function exhibiting the same biological effect, despite thesevariations. Each of these represents a separate embodiment of thispresent invention.

In one embodiment, the formulations and methods of the present inventionmay be used for gene silencing applications. In one embodiment, theactivity or function of a particular gene is suppressed or diminished,via the use of anti-sense oligonucleotides, which are chimericmolecules, containing two or more chemically distinct regions, each madeup of at least one nucleotide.

In one embodiment, chimeric oligonucleotides comprise at least oneregion wherein the oligonucleotide is modified so as to confer upon theoligonucleotide an increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget polynucleotide. An additional region of the oligonucleotide mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids, which according to this aspect of the invention, serves as ameans of gene silencing via degradation of specific sequences. Cleavageof the RNA target can be routinely detected by gel electrophoresis and,if necessary, associated nucleic acid hybridization techniques known inthe art.

The chimeric antisense oligonucleotides may, in one embodiment, beformed as composite structures of two or more oligonucleotides and/ormodified oligonucleotides, as is described in the art (see, for example,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922), and may, in another embodiment, comprise a ribozymesequence.

Inhibition of gene expression, activity or function is effected, inanother embodiment, via the use of small interfering RNAs, whichprovides sequence-specific inhibition of gene expression. Administrationof double stranded/duplex RNA (dsRNA) corresponding to a single gene inan organism can silence expression of the specific gene by rapiddegradation of the mRNA in affected cells. This process is referred toas gene silencing, with the dsRNA functioning as a specific RNAinhibitor (RNAi). RNAi may be derived from natural sources, such as inendogenous virus and transposon activity, or it can be artificiallyintroduced into cells (Elbashir S M, et al (2001). Nature 411:494-498)via microinjection (Fire et al. (1998) Nature 391: 806-11), or bytransformation with gene constructs generating complementary RNAs orfold-back RNA, or by other vectors (Waterhouse, P. M., et al. (1998).Proc. Natl. Acad. Sci. USA 95, 13959-13964 and Wang, Z., et al. (2000).J. Biol. Chem. 275, 40174-40179). The RNAi mediating mRNA degradation,in one embodiment, comprises duplex or double-stranded RNA, or, in otherembodiments, include single-stranded RNA, isolated RNA (partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA), as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion and/or alteration of one or morenucleotides.

In one embodiment, the nucleic acid of the formulations and methods ofthe instant invention encode a therapeutic polypeptide. In oneembodiment, the term “polypeptide” refers to a molecule comprised ofamino acid residues joined by peptide (i.e., amide) bonds and includespeptides, polypeptides, and proteins. Hence, in one embodiment, thepolypeptides of this invention may have single or multiple chains ofcovalently linked amino acids and may further contain intrachain orinterchain linkages comprised of disulfide bonds. In one embodiment,some polypeptides may also form a subunit of a multiunit macromolecularcomplex. In one embodiment, the polypeptides can be expected to possessconformational preferences and to exhibit a three-dimensional structure.Both the conformational preferences and the three-dimensional structurewill usually be defined by the polypeptide's primary (i.e., amino acid)sequence and/or the presence (or absence) of disulfide bonds or othercovalent or non-covalent intrachain or interchain interactions.

In one embodiment, the term “peptide” refers to native peptides (eitherdegradation products, synthetically synthesized peptides or recombinantpeptides) and/or peptidomimetics (typically, synthetically synthesizedpeptides), such as peptoids and semipeptoids which are peptide analogs,which may have, for example, modifications rendering the peptides morestable

while in a body or more capable of penetrating into cells. Suchmodifications include, but are not limited to N terminus modification, Cterminus modification, peptide bond modification, including, but notlimited to, CH₂—NH, CH₂—S, CH₂—S═O, O═C—NH, CH₂—O, CH₂—CH₂, S═C—NH,CH═CH or CF═CH, backbone modifications, and residue modification.Methods for preparing peptidomimetic compounds are well known in the artand are specified, for example, in Quantitative Drug Design, C.A.Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which isincorporated by reference as if fully set forth herein.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH₃)—CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH₂—), aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(—CH₂—NH—), hydroxyethylene bonds (—CH(OH)—CH₂—), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),peptide derivatives (—N(R)—CH₂—CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom. These modifications canoccur at any of the bonds along the peptide chain and even at several(2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic non-natural acid such as TIC, naphthylelanine (Nol),ring-methylated derivatives of Phe, halogenated derivatives of Phe oro-methyl-Tyr. In addition to the above, the peptides of the presentinvention may also include one or more modified amino acids or one ormore non-amino acid monomers (e.g. fatty acids, complex carbohydratesetc).

In one embodiment, the term “amino acid” or “amino acids” is understoodto include the 20 naturally occurring amino acids; those amino acidsoften modified post-translationally in vivo, including, for example,hydroxyproline, phosphoserine and phosphothreonine; and other unusualamino acids including, but not limited to, 2-aminoadipic acid,hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.Furthermore, the term “amino acid” may include both D- and L-aminoacids.

As used herein, the term “amino acid” refers to either the D or Lstereoisomer form of the amino acid, unless otherwise specificallydesignated. Also encompassed within the scope of this invention areequivalent proteins or equivalent peptides, e.g., having the biologicalactivity of purified wild type tumor suppressor protein. “Equivalentproteins” and “equivalent polypeptides” refer to compounds that departfrom the linear sequence of the naturally occurring proteins orpolypeptides, but which have amino acid substitutions that do not changeit's biologically activity. These equivalents can differ from the nativesequences by the replacement of one or more amino acids with relatedamino acids, for example, similarly charged amino acids, or thesubstitution or modification of side chains or functional groups.

The peptides or polypeptides, or the DNA sequences encoding same, may beobtained from a variety of natural or unnatural sources, such as aprokaryotic or a eukaryotic cell. In one embodiment, the source cell maybe wild type, recombinant, or mutant. In another embodiment, theplurality of peptides or polypeptides may be endogenous tomicroorganisms, such as bacteria, yeast, or fungi, to a virus, to ananimal (including mammals, invertebrates, reptiles, birds, and insects)or to a plant cell.

In another embodiment, the peptides or polypeptides may be obtained frommore specific sources, such as the surface coat of a virion particle, aparticular cell lysate, a tissue extract, or they may be restricted tothose polypeptides that are expressed on the surface of a cell membrane.

In another embodiment, the peptide or polypeptide is derived from aparticular cell or tissue type, developmental stage or disease conditionor stage. In one embodiment, the disease condition or stage is cancer,in another embodiment, the disease condition is an infection, which inanother embodiment, is an HIV infection. In another embodiment, thedisease condition is a developmental disorder, while in anotherembodiment, the disease condition is a metabolic disorder.

The polypeptide of the present invention can be of any size. As can beexpected, the polypeptides can exhibit a wide variety of molecularweights, some exceeding 150 to 200 kilodaltons (kD). Typically, thepolypeptides may have a molecular weight ranging from about 5,000 toabout 100,000 daltons. Still others may fall in a narrower range, forexample, about 10,000 to about 75,000 daltons, or about 20,000 to about50,000 daltons. In an alternative embodiment, the polypeptides of thepresent invention may be 1-250 amino acid residues long. In anotherembodiment, the polypeptides of the present invention may be 10-200amino acid residues long. In an alternative embodiment, the polypeptidesof the present invention may be 50-100 amino acid residues long. In analternative embodiment, the polypeptides of the present invention may be1-250 amino acid residues long. In an alternative embodiment, thepolypeptides of the present invention may be 1-250 amino acid residueslong. In one embodiment, the maximum size of the peptide or polypeptideis determined by the vector from which it is expressed, which in oneembodiment, is approximately between 20 and 37 kD, between 20 and 25 kD,between 25 and 30 kD, between 30 and 37 kD, or between 35 and 37 kD. Inanother embodiment, the polypeptide is a 34 kD glycoprotein.

In another embodiment, the peptides or polypeptides are agonists. Inanother embodiment, the peptides or polypeptides are antagonists. Inanother embodiment, the peptides or polypeptides are antigens. Inanother embodiment, the peptides or polypeptides are enzymes. In anotherembodiment, the peptides or polypeptides are activators of enzymes orother substrates. In another embodiment, the peptides or polypeptidesare inhibitors of enzymes or other substrates. In another embodiment,the peptides or polypeptides are hormones. In another embodiment, thepeptides or polypeptides are regulatory proteins. Regulatory proteinscommand the numerous interactions that govern the expression andreplication of genes, the performance of enzymes, the interplay betweencells and their environment, and many other manifestations. In anotherembodiment, the peptides or polypeptides are cytoskeletal proteins.Cytoskeletal proteins form a flexible framework for the cell, provideattachment points for organelles and formed bodies, and makecommunication between parts of the cell possible. In another embodiment,the peptides or polypeptides are toxins. In another embodiment, thetherapeutic nucleic acids of the present invention encode one or moresuicide genes.

In another embodiment, the peptides or polypeptides are functionalfragments of agonists, antagonists, antigens, enzymes, enzymeactivators, enzyme inhibitors, enzyme substrates, hormones, regulatoryproteins, cytoskeletal proteins, or toxins. “Functional fragments” aremeant to indicate a portion of the peptide or polypeptide which iscapable of performing one or more of the functions of the peptide orpolypeptide, even in the absence of the remainder of the peptide orpolypeptide. In one embodiment, the functional fragment is sufficient tomediate an intermolecular interaction with a target of interest.

In an alternative embodiment, the peptide binds DNA or RNA or a fragmentthereof. In one embodiment, the DNA or RNA binding peptide may be any ofthe many known in the art including, but not limited to: Zinc fingerproteins such as Beta-beta-alpha zinc finger proteins, Nuclear receptorproteins, Loop-sheet-helix type protein, and GAL4 type protein; theHelix-turn-helix proteins such as Cro and repressor proteins, Lad purinerepressor proteins (PurR), Fold restriction endonuclease(DNA-recognition region), Gamma-delta recombinase protein (C-terminaldomain), Hin recombinase protein, Trp repressor protein, Diptheria toxrepressor, Catabolite gene activator proteins (CAP), Homeodomainproteins, RAP1 protein, Prd paired protein, Tc3 transposase protein,TFIIB family, Interferon regulatory factor, Transcription factor family,and ETS domain family bacteriophage; and the Leucine zipper proteinssuch as Basic zipper proteins and Zipper-type proteins(helix-loop-helix). In another embodiment, the DNA or RNA bindingpeptide may be other alpha-helix proteins such as Cre recombinasefamily, Papillomavirus-1 E2 protein, Histone family, Ebna 1 nuclearprotein family, Skn-1 transcription factor, High mobility group family,and MADS box family; Beta-sheet proteins such as TATA Box-BindingProteins; Beta-hairpin/ribbon proteins such as Met repressor protein,Tus replication terminator protein, Integration host factor protein,Hyperthermophile DNA binding protein, Arc repressor, Transcriptionfactor T domain; and other protein families such as Rel homology regionproteins and Stat family. In another embodiment, the DNA or RNA bindingpeptide may be enzymes such as Methyl transferase proteins, PvuIIEndonuclease protein, Endonuclease V protein, EcoRV Endonuclease family,BamHI Endonuclease family, EcoRI endonuclease family, DNA mismatchendonuclease, DNA polymerase I protein, DNA polymerase T7, Dnase Iproteins, DNA polymerase beta proteins, Uraci-DNA glycosylase,Methyladenine-DNA glycosylase, Homing endonuclease, and Topoisomerase Ior viral proteins such as HIV reverse transcriptase.

In another embodiment, the peptide or polypeptide is a transcriptionalor translational activator or a fragment thereof. In another embodiment,the peptide or polypeptide is a transcriptional or translationalrepressor or a fragment thereof. In another embodiment, the peptide orpolypeptide is a receptor or a fragment thereof.

In one embodiment, the peptide or polypeptide may represent a cognatepeptide of any of the peptides or polypeptides described hereinabove. A“cognate” peptide is any peptide that interacts and/or binds to anothermolecule.

According to other embodiments of the present invention, recombinantgene products may be encoded by a polynucleotide having a modifiednucleotide sequence, as compared to a corresponding naturalpolynucleotide.

In addition to proteins, recombinant gene products may also comprisefunctional RNA molecules.

According to another embodiment of the present invention, theformulations and methods of the present invention may provide amicro-organ producing functional RNA molecules. Functional RNA moleculesmay comprise antisense oligonucleotide sequences, ribozymes comprisingthe antisense oligonucleotide described herein and a ribozyme sequencefused thereto. Such a ribozyme is readily synthesizable using solidphase oligonucleotide synthesis.

Ribozymes are being increasingly used for the sequence-specificinhibition of gene expression by the cleavage of mRNAs encoding proteinsof interest [Welch et al., “Expression of ribozymes in gene transfersystems to modulate target RNA levels.” Curr Opin Biotechnol. 1998October; 9(5):486-96]. The possibility of designing ribozymes to cleaveany specific target RNA has rendered them valuable tools in both basicresearch and therapeutic applications. In the therapeutics area,ribozymes have been exploited to target viral RNAs in infectiousdiseases, dominant oncogenes in cancers and specific somatic mutationsin genetic disorders [Welch et al., “Ribozyme gene therapy for hepatitisC virus infection.” Clin Diagn Virol. Jul. 15, 1998; 10(2-3):163-71.Most notably, several ribozyme gene therapy protocols for HIV patientsare already in Phase 1 trials. More recently, ribozymes have been usedfor transgenic animal research, gene target validation and pathwayelucidation. Several ribozymes are in various stages of clinical trials.ANGIOZYME was the first chemically synthesized ribozyme to be studied inhuman clinical trials. ANGIOZYME specifically inhibits formation of theVEGF-r (Vascular Endothelial Growth Factor receptor), a key component inthe angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well asother firms has demonstrated the importance of anti-angiogenesistherapeutics in animal models. HEPTAZYME, a ribozyme designed toselectively destroy Hepatitis C Virus (HCV) RNA, was found effective indecreasing Hepatitis C viral RNA in cell culture assays.

As described hereinabove, in one embodiment, the formulations andmethods of the present invention provide a therapeutic formulationcomprising a nucleic acid sequence encoding a therapeutic polypeptide.In one embodiment, the term “therapeutic” refers to a molecule, whichwhen provided to a subject in need, provides a beneficial effect. Insome cases, the molecule is therapeutic in that it functions to replacean absence or diminished presence of such a molecule in a subject. Inone embodiment, the therapeutic protein is that of a protein which isabsent in a subject, such as in cases of subjects with an endogenousnull or mis-sense mutation of a required protein. In other embodiments,the endogenous protein is mutated, and produces a non-functionalprotein, compensated for by the provision of the functional protein. Inother embodiments, expression of a heterologous protein is additive tolow endogenous levels, resulting in cumulative enhanced expression of agiven protein. In other embodiments, the molecule stimulates a signalingcascade that provides for expression, or secretion, or others of acritical element for cellular or host functioning.

In one embodiment, the term “therapeutic formulation” describes asubstance applicable for use in the diagnosis, or in another embodiment,cure, or in another embodiment, mitigation, or in another embodiment,treatment, or in another embodiment, prevention of a disease, disorder,condition or infection. In one embodiment, the “therapeutic formulation”of this invention refers to any substance which affect the structure orfunction of the target to which it is applied.

In another embodiment, the “therapeutic formulation” of the presentinvention is a molecule that alleviates a symptom of a disease ordisorder when administered to a subject afflicted thereof. In oneembodiment, the “therapeutic formulation” of this invention is asynthetic molecule, or in another embodiment, a naturally occurringcompound isolated from a source found in nature.

In one embodiment, the therapeutic polypeptide is erythropoietin, whilein another embodiment, the therapeutic polypeptide is interferon alpha,which in one embodiment, is interferon alpha 2b. In one embodiment, saidtherapeutic polypeptide is any other therapeutic polypeptide.

In one embodiment, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor lessen the targeted pathologic condition or disorder as describedhereinabove. Thus, in one embodiment, treating may include directlyaffecting or curing, suppressing, inhibiting, preventing, reducing theseverity of, delaying the onset of, reducing symptoms associated withthe disease, disorder or condition, or a combination thereof. Thus, inone embodiment, “treating” refers inter alia to delaying progression,expediting remission, inducing remission, augmenting remission, speedingrecovery, increasing efficacy of or decreasing resistance to alternativetherapeutics, or a combination thereof. In one embodiment, “preventing”refers, inter alia, to delaying the onset of symptoms, preventingrelapse to a disease, decreasing the number or frequency of relapseepisodes, increasing latency between symptomatic episodes, or acombination thereof. In one embodiment, “suppressing” or “inhibiting”,refers inter alia to reducing the severity of symptoms, reducing theseverity of an acute episode, reducing the number of symptoms, reducingthe incidence of disease-related symptoms, reducing the latency ofsymptoms, ameliorating symptoms, reducing secondary symptoms, reducingsecondary infections, prolonging patient survival, or a combinationthereof.

In one embodiment, symptoms are primary, while in another embodiment,symptoms are secondary. In one embodiment, “primary” refers to a symptomthat is a direct result of a particular disease, while in oneembodiment; “secondary” refers to a symptom that is derived from orconsequent to a primary cause. In one embodiment, the compounds for usein the present invention treat primary or secondary symptoms orsecondary complications related to said disease. In another embodiment,“symptoms” may be any manifestation of a disease or pathologicalcondition.

In one embodiment, a therapeutic nucleic acid may encode a therapeuticpolypeptide, which may in one embodiment, comprise an enzyme, an enzymecofactor, a cytotoxic protein, an antibody, a channel protein, atransporter protein, a growth factor, a hormone, a cytokine, a receptor,a mucin, a surfactant, an aptamer or a hormone. In another embodiment,the therapeutic polypeptide may be of one or more of the categories asdescribed above. In another embodiment, a therapeutic nucleic acid mayencode functional RNA as described hereinbelow.

In one embodiment, the term “antibody or antibody fragment” refers tointact antibody molecules as well as functional fragments thereof, suchas Fab, F(ab′)2, and Fv that are capable of binding to an epitope. Inone embodiment, an Fab fragment refers to the fragment which contains amonovalent antigen-binding fragment of an antibody molecule, which canbe produced by digestion of whole antibody with the enzyme papain toyield an intact light chain and a portion of one heavy chain. In oneembodiment, Fab′ fragment refers to a part of an antibody molecule thatcan be obtained by treating whole antibody with pepsin, followed byreduction, to yield an intact light chain and a portion of the heavychain. Two Fab′ fragments may be obtained per antibody molecule. In oneembodiment, (Fab′)₂ refers to a fragment of an antibody that can beobtained by treating whole antibody with the enzyme pepsin withoutsubsequent reduction. In another embodiment, F(ab′)₂ is a dimer of twoFab′ fragments held together by two disulfide bonds. In one embodiment,Fv, may refer to a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains. In one embodiment, the antibody fragmentmay be a single chain antibody (“SCA”), a genetically engineeredmolecule containing the variable region of the light chain and thevariable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, 1988, incorporated herein by reference).

In one embodiment, the antibody will recognize an epitope, which inanother embodiment, refers to antigenic determinant on an antigen towhich the paratope of an antibody binds. Epitopic determinants may, inother embodiments, consist of chemically active surface groupings ofmolecules such as amino acids or carbohydrate side chains and in otherembodiments, may have specific three dimensional structuralcharacteristics, and/or in other embodiments, have specific chargecharacteristics.

In one embodiment, the epitope recognized is from a pathogen, or inanother embodiment, a pathogenic cell, or in another embodiment, aprotein aberrantly expressed, which, in another embodiment, may refer tothe location, quantity, or combination thereof of expression.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry,Methods, 2: 106-10, 1991.

In one embodiment, the antibody is tumoricidal, and is therebytherapeutic in certain cancers. Antibodies that possess tumoricidalactivity are also known in the art, the use of any of which mayrepresent an embodiment of this invention, including IMC-C225, EMD72000, OvaRex Mab B43.13, anti-ganglioside G(D2) antibody ch14.18,C017-1A, trastuzumab, rhuMAb VEGF, sc-321, AF349, BAF349, AF743, BAF743,MAB743, AB1875, Anti-Flt-4AB3127, FLT41-A, rituximab, 2C3, CAMPATH 1H,2G7, Alpha IR-3, ABX-EGF, MDX-447, anti-p75 IL-2R, anti-p64 IL-2R, and2A11.

In one embodiment, the “therapeutic nucleic acid” of this invention mayencode or the “therapeutic polypeptide” may be molecules that serve asantihypertensives, antidepressants, antianxiety agents, anticlottingagents, anticonvulsants, blood glucose-lowering agents, decongestants,antihistamines, antitussives, anti-inflammatories, antipsychotic agents,cognitive enhancers, cholesterol-reducing agents, antiobesity agents,autoimmune disorder agents, anti-impotence agents, antibacterial andantifungal agents, hypnotic agents, anti-Parkinsonism agents,antibiotics, antiviral agents, anti-neoplastics, barbituates, sedatives,nutritional agents, beta blockers, emetics, anti-emetics, diuretics,anticoagulants, cardiotonics, androgens, corticoids, anabolic agents,growth hormone secretagogues, anti-infective agents, coronaryvasodilators, carbonic anhydrase inhibitors, antiprotozoals,gastrointestinal agents, serotonin antagonists, anesthetics,hypoglycemic agents, dopaminergic agents, anti-Alzheimer's Diseaseagents, anti-ulcer agents, platelet inhibitors and glycogenphosphorylase inhibitors.

In one embodiment, the “therapeutic formulation” of this invention isantibacterial, antiviral, antifungal or antiparasitic. In anotherembodiment, the therapeutic formulation has cytotoxic or anti-canceractivity. In another embodiment, the therapeutic formulation isimmunostimulatory. In another embodiment, the therapeutic formulationinhibits inflammatory or immune responses.

In one embodiment, the therapeutic nucleic acids may encode or thetherapeutic polypeptides may be cytokines, such as interferons orinterleukins, or their receptors. Lack of expression of cytokines, or ofthe appropriate ones, has been implicated in susceptibility to diseases,and enhanced expression may lead to resistance to a number ofinfections. Expression patterns of cytokines may be altered to produce abeneficial effect, such as for example, a biasing of the immune responsetoward a Th1 type expression pattern, or a Th2 pattern in infection, orin autoimmune disease, wherein altered expression patterns may provebeneficial to the host.

In another embodiment, the therapeutic nucleic acid may encode or thetherapeutic polypeptide may be an enzyme, such as one involved inglycogen storage or breakdown. In another embodiment, the therapeuticprotein comprises a transporter, such as an ion transporter, for exampleCFTR, or a glucose transporter, or other transporters whose deficiency,or inappropriate expression, results in a variety of diseases.

In another embodiment, the therapeutic nucleic acid encodes or thetherapeutic polypeptide is a tumor suppressor or pro-apoptotic compound,which alters progression of cancer-related events.

In another embodiment, the therapeutic nucleic acid of the presentinvention may encode or the therapeutic polypeptide may be animmunomodulating protein. In one embodiment, the immunomodulatingprotein comprises cytokines, chemokines, complement or components, suchas interleukins 1 to 15, interferons alpha, beta or gamma, tumournecrosis factor, granulocyte-macrophage colony stimulating factor(GM-CSF), macrophage colony stimulating factor (M-CSF), granulocytecolony stimulating factor (G-CSF), chemokines such as neutrophilactivating protein (NAP), macrophage chemoattractant and activatingfactor (MCAF), RANTES, macrophage inflammatory peptides MIP-1a andMIP-1b, or complement components.

In another embodiment, a therapeutic nucleic acid of this invention mayencode or a therapeutic polypeptide may be a growth factor, ortissue-promoting factor. In one embodiment, the therapeutic compound isa bone morphogenetic protein, or OP-1, OP-2, BMP-5, BMP-6, BMP-2, BMP-3,BMP-4, BMP-9, DPP, Vg-1, 60A, or Vgr-1. In another embodiment, thetherapeutic nucleic acid encodes an RNA or peptide that facilitatesnerve regeneration or repair, and may include NGF, or other growthfactors. In another embodiment, the therapeutic polypeptide facilitatesnerve regeneration or repair, and may include NGF, or other growthfactors.

In another embodiment, the therapeutic nucleic acid may encode or thetherapeutic polypeptide may be natural or non-natural insulins,amylases, proteases, lipases, kinases, phosphatases, glycosyltransferases, trypsinogen, chymotrypsinogen, carboxypeptidases,hormones, ribonucleases, deoxyribonucleases, triacylglycerol lipase,phospholipase A2, elastases, amylases, blood clotting factors, UDPglucuronyl transferases, ornithine transcarbamoylases, cytochrome p450enzymes, adenosine deaminases, serum thymic factors, thymic humoralfactors, thymopoietins, growth hormones, somatomedins, costimulatoryfactors, antibodies, colony stimulating factors, erythropoietin,epidermal growth factors, hepatic erythropoietic factors(hepatopoietin), liver-cell growth factors, interleukins, interferons,negative growth factors, fibroblast growth factors, transforming growthfactors of the a family, transforming growth factors of the 0 family,gastrins, secretins, cholecystokinins, somatostatins, serotonins,substance P, transcription factors or combinations thereof.

In another embodiment, the gene comprises a reporter gene. In oneembodiment, the reporter gene encodes a fluorescent protein. In oneembodiment, the fluorescent protein is yECitrine or a yellow fluorescentprotein. In one embodiment, the fluorescent protein is the jellyfishgreen fluorescent protein, or a mutant or variant thereof. In anotherembodiment, the GMMOs specifically may comprise any gene other than areporter gene or a gene encoding a reporter protein.

In another embodiment, the reporter gene confers drug resistance. In oneembodiment, the reporter gene confers resistance to an antibiotic, suchas, for example, ampicilin, kanamycin, tetracycline, or others, as willbe appreciated by one skilled in the art. In another embodiment, theantibiotic resistance genes may include those conferring resistance toneomycin (neo), blasticidin, spectinomycin, erythromycin, phleomycin,Tn917, gentamycin, and bleomycin. An example of the neomycin resistancegene is the neomycin resistance gene of transposon Tn5 that encodes forneomycin phosphotransferase 11, which confers resistance to variousantibiotics, including G418 and kanamycin. In another embodiment, thereporter is a chloramphenicol acetyl transferase gene (cat) and confersresistance to chloramphenicol.

In one embodiment, the formulations and methods of this invention arefor prevention of, or therapeutic intervention of viral infection, or inanother embodiment, bacterial, parasitic, or fungal infection, or acombination thereof.

According to this aspect of the invention, the formulations and methodsof this invention are for prevention of, or therapeutic intervention indisease. In one embodiment, the disease for which the subject is thustreated may comprise, but is not limited to: muscular dystrophy, cancer,cardiovascular disease, hypertension, infection, renal disease,neurodegenerative disease, such as alzheimer's disease, parkinson'sdisease, huntington's chorea, Creurtfeld-Jacob disease, autoimmunedisease, such as lupus, rheumatoid arthritis, endocarditis, Graves'disease or ALD, respiratory disease such as asthma or cystic fibrosis,bone disease, such as osteoporosis, joint disease, liver disease,disease of the skin, such as psoriasis or eczema, ophthalmic disease,otolaryngeal disease, other neurological disease such as Turretsyndrome, schizophrenia, depression, autism, or stoke, or metabolicdisease such as a glycogen storage disease or diabetes. It is to beunderstood that any disease whereby expression of a particular protein,provision of a therapeutic protein, provision of a drug, inhibition ofexpression of a particular protein, etc., which can be accomplished viathe formulations of this invention and according to the methods of thisinvention, is to be considered as part of this invention.

In one embodiment, the formulations and methods of the instant inventioncomprise a nucleic acid sequence operably linked to one or moreregulatory sequences. In one embodiment, a nucleic acid moleculeintroduced into a cell of a micro-organ is in a form suitable forexpression in the cell of the gene product encoded by the nucleic acid.Accordingly, in one embodiment, the nucleic acid molecule includescoding and regulatory sequences required for transcription of a gene (orportion thereof). When the gene product is a protein or peptide, thenucleic acid molecule includes coding and regulatory sequences requiredfor translation of the nucleic acid molecule include promoters,enhancers, polyadenylation signals, sequences necessary for transport ofan encoded protein or peptide, for example N-terminal signal sequencesfor transport of proteins or peptides to the surface of the cell orsecretion, in one embodiment.

Nucleotide sequences which regulate expression of a gene product (e.g.,promoter and enhancer sequences) are selected based upon the type ofcell in which the gene product is to be expressed and the desired levelof expression of the gene product. For example, a promoter known toconfer cell-type specific expression of a gene linked to the promotercan be used. A promoter specific for myoblast gene expression can belinked to a gene of interest to confer muscle-specific expression ofthat gene product. Muscle-specific regulatory elements which are knownin the art include upstream regions from the dystrophin gene (Klamut etal., (1989) Mol. Cell. Biol. 0.9:2396), the creatine kinase gene (Buskinand Hauschka, (1989) Mol. Cell. Biol. 9:2627) and the troponin gene (Marand Ordahl, (1988) Proc. Natl. Acad. Sci. USA. 85:6404). Negativeresponse elements in keratin genes mediate transcriptional repression(Jho Sh et al, (2001). J. Biol Chem). Regulatory elements specific forother cell types are known in the art (e.g., the albumin enhancer forliver-specific expression; insulin regulatory elements for pancreaticislet cell-specific expression; various neural cell-specific regulatoryelements, including neural dystrophin, neural enolase and A4 amyloidpromoters). Alternatively, a regulatory element which can directconstitutive expression of a gene in a variety of different cell types,such as a viral regulatory element, can be used. Examples of viralpromoters commonly used to drive gene expression include those derivedfrom polyoma virus, Adenovirus 2, cytomegalovirus (CMV) and Simian Virus40, and retroviral LTRs. Alternatively, a regulatory element whichprovides inducible expression of a gene linked thereto can be used. Theuse of an inducible regulatory element (e.g., an inducible promoter)allows for modulation of the production of the gene product in the cell.Examples of potentially useful inducible regulatory systems for use ineukaryotic cells include hormone-regulated elements (e.g., see Mader, S,and White, J. H. (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607),synthetic ligand-regulated elements (see, e.g., Spencer, D. M. et al1993) Science 262:1019-1024) and ionizing radiation-regulated elements(e.g., see Manome, Y. Et al. (1993) Biochemistry 32:10607-10613; Datta,R. et al. (1992) Proc. Natl. Acad. Sci. USA89:1014-10153). Additionaltissue-specific or inducible regulatory systems which may be developedcan also be used in accordance with the invention.

In one embodiment, a regulatory sequence of the instant invention maycomprise a CMV promoter, while in another embodiment; the regulatorysequence may comprise a CAG promoter. In one embodiment, a CAG promoteris a composite promoter that combines the human cytomegalovirusimmediate-early enhancer and a modified chicken beta-actin promoter andfirst intron. In one embodiment, a regulatory sequence may comprise asimian virus (SV)-40 polyadenylation sequence, which in one embodiment,is the mechanism by which most messenger RNA molecules are terminated attheir 3′ ends in eukaryotes. In one embodiment, the polyadenosine(poly-A) tail protects the mRNA molecule from exonucleases and isimportant for transcription termination, for export of the mRNA from thenucleus, and for translation. In another embodiment, a formulation ofthe present invention may comprise one or more regulatory sequences.

In one embodiment, formulations of the instant invention comprising CMVor CAG promoters in conjunction with SV40 demonstrate long-term, high invitro (FIGS. 1, 5, and 7B) and in vivo (FIG. 6A) expression levels ofEPO and IFN-alpha. Without being bound by theory, one factor that maycontribute to the long-lasting, high levels of gene product frommicro-organs of the instant invention is the use of CMV, oralternatively, CAG as a promoter, which may be especially effective inmicro-organ explants in promoting constitutive gene expression.

In one embodiment, the term “promoter” refers to a DNA sequence, which,in one embodiment, is directly upstream of the coding sequence and isimportant for basal and/or regulated transcription of a gene. In oneembodiment, a promoter of the present invention is operatively linked toa gene of interest. In another embodiment, the promoter is a mutant ofthe endogenous promoter, which is normally associated with expression ofthe gene of interest, under the appropriate conditions.

In one embodiment, a promoter of the compositions and for use in themethods of the present invention is a regulatable promoter. In anotherembodiment, a regulatable promoter refers to a promoter wherebyexpression of a gene downstream occurs as a function of the occurrenceor provision of specific conditions which stimulate expression from theparticular promoter. In some embodiments, such conditions result indirectly turning on expression, or in other embodiments, removeimpediments to expression. In some embodiments, such conditions resultin turning off, or reducing expression.

In one embodiment, such conditions may comprise specific temperatures,nutrients, absence of nutrients, presence of metals, or other stimuli orenvironmental factors as will be known to one skilled in the art. In oneembodiment, a regulatable promoter may be regulated by galactose (e.g.UDP-galactose epimerase (GAL10), galactokinase (GAL1)), glucose (e.g.alcohol dehydrogenase II (ADH2)), or phosphate (e.g. acid phosphatase(PHO5)). In another embodiment, a regulatable promoter may be activatedby heat shock (heat shock promoter) or chemicals such as IPTG orTetracycline, or others, as will be known to one skilled in the art. Itis to be understood that any regulatable promoter, and conditions forsuch regulation is encompassed by the vectors, nucleic acids and methodsof this invention, and represents an embodiment thereof.

In one embodiment, the formulations and methods of the instant inventionincrease the levels of a therapeutic polypeptide or nucleic acid by atleast 5% over basal levels. In another embodiment, the levels of atherapeutic polypeptide or nucleic acid are increased by at least 7%, inanother embodiment, by at least 10%, in another embodiment, by at least15%, in another embodiment, by at least 20%, in another embodiment, byat least 25%, in another embodiment, by at least 30%, in anotherembodiment, by at least 40%, in another embodiment, by at least 50%, inanother embodiment, by at least 60%, in another embodiment, by at least75%, in another embodiment, by at least 100%, in another embodiment, byat least 125%, in another embodiment, by at least 150% over basallevels, in another embodiment, by at least 200% over basal levels.

In one embodiment, expression of a therapeutic polypeptide or nucleicacid via the formulation of the present invention is increased comparedto “basal levels”, which in one embodiment, are levels of the geneexpressed in hosts or cell culture that had not been administered orotherwise contacted with the therapeutic formulation of the presentinvention.

In another embodiment, the formulations and methods of the instantinvention increase the levels of a therapeutic polypeptide or nucleicacid to approximately 2000 ng/day, or in another embodiment, 1500ng/day, or in another embodiment, 1000 ng/day, or in another embodiment,750 ng/day, or in another embodiment, 500 ng/day, or in anotherembodiment, 250 ng/day, or in another embodiment, 150 ng/day, or inanother embodiment, 100 ng/day, or in another embodiment, 75 ng/day, orin another embodiment, 50 ng/day, or in another embodiment, 25 ng/day.In another embodiment, the formulations and methods of the instantinvention increase the levels of a therapeutic polypeptide to between20-70 mU/mL, or in another embodiment, 50-100 mU/mL, or in anotherembodiment, 5-20 mU/mL, or in another embodiment, 100-200 mU/mL, or inanother embodiment, 10-70 mU/mL, or in another embodiment, 5-80 mU/mL.In another embodiment, the formulations and methods of the instantinvention increase the levels of a therapeutic polypeptide to between500-1000 mU/mL, or in another embodiment, 250-750 mU/mL, or in anotherembodiment, 500-5000 mU/mL.

In one embodiment, the formulations and methods of the instant inventionincrease the levels of a functional marker, which in one embodiment, ishematocrit levels, by at least 5% over basal levels. In anotherembodiment, the levels of the functional marker are increased by atleast 7%, in another embodiment, by at least 10%, in another embodiment,by at least 15%, in another embodiment, by at least 20%, in anotherembodiment, by at least 25%, in another embodiment, by at least 30%, inanother embodiment, by at least 40%, in another embodiment, by at least50%, in another embodiment, by at least 60%, in another embodiment, byat least 75%, in another embodiment, by at least 100%, in anotherembodiment, by at least 125%, in another embodiment, by at least 150%over basal levels, in another embodiment, by at least 200% over basallevels.

In one embodiment, the therapeutic formulation of the present inventionis “long-lasting”, which in one embodiment refers to a formulation thatcan increase secretion, expression, production, circulation orpersistence of a therapeutic polypeptide or nucleic acid. In oneembodiment, expression levels of a therapeutic polypeptide or nucleicacid are increased over basal levels for at least one month, or inanother embodiment, for at least six months. In another embodiment, thelevels of hematocrit are increased for at least 2 weeks, in anotherembodiment, for at least 3 weeks, in another embodiment, for at least 4weeks, in another embodiment, for at least 5 weeks, in anotherembodiment, for at least 6 weeks, in another embodiment, for at least 8weeks, in another embodiment, for at least 2 months, in anotherembodiment, for at least 2 months in another embodiment, for at least 2months in another embodiment, for at least 3 months in anotherembodiment, for at least 4 months, in another embodiment, for at least 5months, in another embodiment, for at least 7 months, in anotherembodiment, for at least 8 months, in another embodiment, for at least 9months, in another embodiment, for at least 10 months, in anotherembodiment, for at least 11 months, or, in another embodiment, for atleast 1 year. In another embodiment, expression levels of a therapeuticpolypeptide or nucleic acid are increased for at least 4-6 months.

In one embodiment, the nucleic acid sequence encoding a therapeuticpolypeptide or nucleic acid is optimized for increased levels oftherapeutic polypeptide or nucleic acid expression, or, in anotherembodiment, for increased duration of therapeutic polypeptide or nucleicacid expression, or, in another embodiment, a combination thereof.

In one embodiment, the term “optimized” refers to a desired change,which, in one embodiment, is a change in gene expression and, in anotherembodiment, in protein expression. In one embodiment, optimized geneexpression is optimized regulation of gene expression. In anotherembodiment, optimized gene expression is an increase in gene expression.According to this aspect and in one embodiment, a 2-fold through1000-fold increase in gene expression compared to wild-type iscontemplated. In another embodiment, a 2-fold to 500-fold increase ingene expression, in another embodiment, a 2-fold to 100-fold increase ingene expression, in another embodiment, a 2-fold to 50-fold increase ingene expression, in another embodiment, a 2-fold to 20-fold increase ingene expression, in another embodiment, a 2-fold to 10-fold increase ingene expression, in another embodiment, a 3-fold to 5-fold increase ingene expression is contemplated.

In another embodiment, optimized gene expression may be an increase ingene expression under particular environmental conditions. In anotherembodiment, optimized gene expression may comprise a decrease in geneexpression, which, in one embodiment, may be only under particularenvironmental conditions.

In another embodiment, optimized gene expression is an increasedduration of gene expression. According to this aspect and in oneembodiment, a 2-fold through 1000-fold increase in the duration of geneexpression compared to wild-type is contemplated. In another embodiment,a 2-fold to 500-fold increase in the duration of gene expression, inanother embodiment, a 2-fold to 100-fold increase in the duration ofgene expression, in another embodiment, a 2-fold to 50-fold increase inthe duration of gene expression, in another embodiment, a 2-fold to20-fold increase in the duration of gene expression, in anotherembodiment, a 2-fold to 10-fold increase in the duration of geneexpression, in another embodiment, a 3-fold to 5-fold increase in theduration of gene expression is contemplated. In another embodiment, theincreased duration of gene expression is compared to gene expression innon-vector-expressing controls, or alternatively, compared to geneexpression in wild-type-vector-expressing controls.

Expression in mammalian cells is hampered, in one embodiment, bytranscriptional silencing, low mRNA half-life, alternative splicingevents, premature polyadenylation, inefficient nuclear translocation andavailability of rare tRNAs pools. The source of many problems inmammalian expressions are found within the message encoding thetransgene including in the autologous expression of many crucialmammalian genes as well. The optimization of mammalian RNAs may includemodification of cis acting elements, adaptation of its GC-content,modifying codon bias with respect to non-limiting tRNAs pools of themammalian cell, avoiding internal homologous regions and excludingRNAi's.

Therefore, in one embodiment, when relying on carefully designedsynthetic genes, stable messages with prolonged half-lives, constitutivenuclear export and high level protein production within the mammalianhost can be expected.

Thus, in one embodiment, optimizing a gene entails adapting the codonusage to the codon bias of host genes, which in one embodiment, are Homosapiens genes; adjusting regions of very high (>80%) or very low (<30%)GC content; avoiding one or more of the following cis-acting sequencemotifs: internal TATA-boxes, chi-sites and ribosomal entry sites;AT-rich or GC-rich sequence stretches; ARE, INS, CRS sequence elements;repeat sequences and RNA secondary structures; (cryptic) splice donorand acceptor sites, branch points; or a combination thereof. In oneembodiment, a gene is optimized for expression in homo sapien cells. Inanother embodiment, a gene is optimized for expression in micro-organs.In another embodiment, a gene is optimized for expression in dermalcells.

In one embodiment, as demonstrated herein, optimized genes, such as EPO,maintain an increase percent of peak expression levels for an extendedperiod of time compared to both non-optimized EPO expressed from agutless adenovirus vector or non-optimized EPO expressed from anadenovirus 5 vector (FIGS. 3 and 4).

In one embodiment, the term “gene” refers to a nucleic acid fragmentthat is capable of being expressed as a specific protein, includingregulatory sequences preceding (5′ non-coding sequences) and following(3′ non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. “Endogenousgene” refers to a native gene in its natural location in the genome ofan organism. A “foreign” gene refers to a gene not normally found in thehost organism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure.

In one embodiment, the therapeutic nucleic acid may be any gene whichencodes an RNA molecule (sense or antisense), peptide, polypeptide,glycoprotein, lipoprotein or combination thereof or to any other postmodified polypeptide. In one embodiment of the invention, the gene ofinterest may be naturally expressed in the tissue sample. In anotherembodiment of this invention, the tissue sample may be geneticallyengineered so that at least one cell will express the gene of interest,which is either not naturally expressed by the cell or has an alteredexpression profile within the cell. In one embodiment, the therapeuticnucleic acid of the present invention may encode or the therapeuticpolypeptide may be any of the proteins listed in U.S. patent applicationSer. No. 10/376,506, which is incorporated herein by reference in itsentirety.

In one embodiment, the genetically modified micro-organ is a geneticallymodified dermal micro-organ. “Dermal” micro-organs may comprise aplurality of dermis components, where in one embodiment; dermis is theportion of the skin located below the epidermis. These components maycomprise skin fibroblast, epithelial cells, other cell types, bases ofhair follicles, nerve endings, sweat and sebaceous glands, and blood andlymph vessels. In one embodiment, a dermal micro-organ may comprise fattissue, wherein in another embodiment, a dermal micro-organ may notcomprise fat tissue. Further details regarding dermal micro-organs,including methods of harvesting, maintaining in culture, and implantingsaid dermal micro-organs, are described in PCT Patent ApplicationWO2004/099363, which is incorporated herein by reference in itsentirety.

In another embodiment, the invention provides a method of providing atherapeutic polypeptide to a subject in need over a sustained periodcomprising providing one or more genetically modified micro-organs, saidmicro-organs comprising a vector comprising a nucleic acid sequenceoperably linked to one or more regulatory sequences; and implanting saidgenetically modified micro-organ in said subject, wherein said nucleicacid sequence encodes a therapeutic polypeptide and whereby theexpression level of the therapeutic nucleic acid or polypeptide isincreased by more than 5% over basal level and said increase ismaintained for greater than one month. In another embodiment, theinvention provides a method of providing a therapeutic polypeptide to asubject in need over a sustained period comprising providing one or moregenetically modified micro-organs, said micro-organs comprising a vectorcomprising a nucleic acid sequence operably linked to one or moreregulatory sequences; and implanting said genetically modifiedmicro-organ in said subject, wherein said nucleic acid sequence encodesa therapeutic polypeptide and wherein said vector is a helper-dependentadenovirus vector. In another embodiment, the invention provides amethod of providing a therapeutic polypeptide to a subject in need overa sustained period comprising providing one or more genetically modifiedmicro-organs, said micro-organs comprising a vector comprising a nucleicacid sequence operably linked to one or more regulatory sequences; andimplanting said genetically modified micro-organ in said subject,wherein said nucleic acid sequence encodes a therapeutic polypeptide andwherein said vector is a helper-dependent adenovirus vector.

In another embodiment, the methods described hereinabove provide atherapeutic nucleic acid to a subject in need wherein the expressionlevel of the therapeutic nucleic acid or polypeptide is increased bymore than 5% over basal level and said increase is maintained forgreater than one hour, 3 hours, 6 hours, 9 hours, 12 hours, 18 hours, 1day, or 2 days, wherein said vector is a helper-dependent adenovirusvector, or a combination thereof.

In one embodiment, this invention provides a therapeutic formulation asdescribed hereinabove in which the therapeutic polypeptide iserythropoietin or wherein the therapeutic nucleic acid encodeserythropoietin. In another embodiment, this invention provides along-lasting erythropoietin formulation comprising a geneticallymodified micro-organ, said micro-organ comprising a vector comprising anucleic acid sequence operably linked to one or more regulatorysequences, wherein said nucleic acid sequence encodes erythropoietin andwhereby said formulation increases erythropoietin levels by more than 5%over basal levels and said increased erythropoietin levels persist forgreater than one month. In another embodiment, the invention provides amethod of providing a therapeutic formulation to a subject in need inwhich the therapeutic polypeptide is erythropoietin or wherein thetherapeutic nucleic acid encodes erythropoietin. In another embodiment,the invention provides a method of providing erythropoietin to a subjectin need.

In another embodiment, this invention provides a method of deliveringerythropoietin to a subject in need over a sustained period comprising:providing one or more genetically modified micro-organs, saidmicro-organs comprising a vector comprising a nucleic acid sequenceoperably linked to one or more regulatory sequences; and implanting saidgenetically modified micro-organ in said subject, wherein said nucleicacid sequence encodes erythropoietin and whereby erythropoietin levelsare increased by more than 5% over basal levels and said increasederythropoietin levels persist for greater than one month.

In another embodiment, this invention provides a method of inducingformation of new blood cells in a subject in need over a sustainedperiod comprising: providing one or more genetically modifiedmicro-organs, said micro-organs comprising a vector comprising a nucleicacid sequence operably linked to one or more regulatory sequences; andimplanting said genetically modified micro-organ in said subject,wherein said nucleic acid sequence encodes erythropoietin and wherebyerythropoietin levels are increased by more than 5% over basal levelsand said increased erythropoietin levels persist for greater than onemonth.

In one embodiment, erythropoietin (EPO) is a glycoprotein hormoneinvolved in the maturation of erythroid progenitor cells intoerythrocytes. In one embodiment, erythropoietin is essential inregulating levels of red blood cells in circulation. Naturally occurringerythropoietin is produced by the kidneys and liver, circulates in theblood, and stimulates the production of red blood cells in bone marrow,in one embodiment, in response to hypoxia.

In one embodiment, EPO of the compositions and methods of the instantinvention may comprise glycosylation patterns similar to those of EPOextracted from human or animal urine, or in another embodiment, plasma.

The identification, cloning, and expression of genes encodingerythropoietin are described in U.S. Pat. Nos. 5,756,349; 5,955,422;5,618,698; 5,547,933; 5,621,080; 5,441,868; and 4,703,008, which areincorporated herein by reference. A description of the purification ofrecombinant erythropoietin from cell medium that supported the growth ofmammalian cells containing recombinant erythropoietin plasmids forexample, are included in U.S. Pat. No. 4,667,016 to Lai et al, which isincorporated herein by reference. Recombinant erythropoietin produced bygenetic engineering techniques involving the expression of a proteinproduct in vitro from a host cell transformed with the gene encodingerythropoietin has been used to treat anemia resulting from chronicrenal failure. Currently, EPO is used in the treatment of anemia ofrenal failure, the anemia associated with HIV infection in zidovudine(AZT) treated patients, and anemia associated with cancer chemotherapy.Administration of rhu-EPO has become routine in the treatment of anemiasecondary to renal insufficiency, where doses of 50-75 u/kg given threetimes per week are used to gradually restore hematocrit and eliminatetransfusion dependency.

Many cell surface and secretory proteins produced by eukaryotic cellsare modified with one or more oligosaccharide groups calledglycosylation, which can dramatically affect protein stability,secretion, and subcellular localization as well as biological activity.In one embodiment, both human urinary derived erythropoietin andrecombinant erythropoietin (expressed in mammalian cells) having theamino acid sequence 1-165 of human erythropoietin comprise threeN-linked and one O-linked oligosaccharide chains which together compriseabout 40% of the total molecular weight of the glycoprotein. In oneembodiment, non-glycosylated erythropoietin has greatly reduced in vivoactivity compared to the glycosylated form but does retain some in vitroactivity. In one embodiment, the EPO of the compositions and for use inthe methods of the present invention are fully glycosylated, while inanother embodiment, they are comprise some glycosylated residues, whilein another embodiment, they are not glycosylated.

In one embodiment, the EPO gene may be a wild-type EPO gene, while inanother embodiment, the EPO gene may be modified. In one embodiment, themodified EPO gene may be optimized.

In one embodiment, the EPO gene has a nucleic acid sequence thatcorresponds to that set forth in Genbank Accession Nos: X02158;AF202312; AF202311; AF202309; AF202310; AF053356; AF202306; AF202307; orAF202308 or encodes a protein sequence that corresponds to that setforth in Genbank Accession Nos: CAA26095; AAF23134; AAF17572; AAF23133;AAC78791; or AAF23132. In another embodiment, the EPO precursor gene hasa nucleic acid sequence that corresponds to that set forth in GenbankAccession Nos: NM_(—)000799; M11319; BC093628; or BC111937 or encodes aprotein sequence that corresponds to that set forth in Genbank AccessionNos: NP_(—)000790; AAA52400; AAH93628; or AAI11938. In anotherembodiment, the EPO gene has a nucleic acid sequence as presented in SEQID No: 7, while in another embodiment, the EPO gene has an amino acidsequence as presented in SEQ ID No: 8. In another embodiment, the EPOgene has a nucleic acid that is homologous to that presented in SEQ IDNo: 7, while in another embodiment, the EPO gene has an amino acidsequence that is homologous to that presented in SEQ ID No: 8.

In one embodiment, the formulations of the present invention may be usedto treat a subject having anemia. In one embodiment, anemia is definedas “a pathologic deficiency in the amount of oxygen-carrying hemoglobinin the red blood cells.” Symptoms of anemia include fatigue, diminishedability to perform daily functions, impaired cognitive function,headache, dizziness, chest pain and shortness of breath, nausea,depression, pain, or a combination thereof. In one embodiment, anemia isassociated with a poorer prognosis and increased mortality.

Anemia is often a consequence of renal failure due to decreasedproduction of erythropoietin from the kidney. In another embodiment,anemia is caused by lowered red blood cell (erythroid) production bybone marrow due to cancer infiltration, lymphoma or leukemia, or marrowreplacement. Other causes of anemia comprise, blood loss due toexcessive bleeding such as hemorrhages or abnormal menstrual bleeding;cancer therapies, such as surgery, radiotherapy, chemotherapy,immunotherapy, or a combination thereof; infiltration or replacement ofcancerous bone marrow; increased hemolysis, which in one embodiment isbreakdown or destruction of red blood cells; low levels oferythropoietin, or a combination thereof. In one embodiment, anemiarefers to Fanconi anemia, which in one embodiment, is an inheritedanemia that leads to bone marrow failure (aplastic anemia) and often toacute myelogenous leukemia (AML). In another embodiment, anemia refersto Diamond Blackfan anemia, normocytic anemia, aplastic anemia,iron-deficiency anemia, vitamin deficiency anemia, Sideroblastic Anemia,Paroxysmal Nocturnal Hemoglobinuria, Anemia of Chronic Disease, Anemiain Kidney Disease and Dialysis, or a combination thereof. In anotherembodiment, the long-lasting EPO formulation of the instant invention isused for treating a diabetic subject. According to this aspect and inone embodiment, the EPO formulation of the instant invention may be usedin conjunction with other treatments for diabetes known in the Art,including, inter alia, insulin administration, oral hypoglycemic drugs,which in one embodiment are sulfonurea drugs, which in one embodimentincluding inter alia glucotrol, glyburide, glynase and amaryl;glucophage, thiazolidinediones including inter alia rezulin, actos andavandia; or a combination thereof. In another embodiment, thelong-lasting EPO formulation of the instant invention is used fortreating a subject suffering from chronic kidney disease, while inanother embodiment, is used for treating a subject suffering fromend-stage renal disease. In another embodiment, the formulations of theinstant invention are used for subjects that are susceptible to theabove-mentioned diseases or conditions.

It is to be understood that the formulations and methods of thisinvention may be used to treat anemia, regardless of the cause of anemiaand whether or not the cause of anemia is known.

In one embodiment, the formulations and method of the present inventionmay be administered with other treatments that are effective in treatinganemia. In one embodiment, other treatments include iron supplements,vitamin B12 supplements, additional sources of erythropoietin,androgens, growth factors such as G-CSF, or a combination thereof. Inanother embodiment, the formulations and method of the present inventionmay be administered in conjunction with other treatments such as bloodand marrow stem cell transplants.

In one embodiment, this invention provides a therapeutic formulation asdescribed hereinabove in which the therapeutic polypeptide is interferonor in which the therapeutic nucleic acid encodes interferon, which inone embodiment, is interferon alpha, which in one embodiment, isinterferon alpha 2a. In another embodiment, the present inventionprovides a long-lasting interferon-alpha formulation comprising agenetically modified micro-organ, said micro-organ comprising a vectorcomprising a nucleic acid sequence operably linked to one or moreregulatory sequences, wherein said nucleic acid sequence encodesinterferon-alpha and whereby said formulation increases interferon-alphalevels by more than 5% over basal levels and said increasedinterferon-alpha levels persist for greater than one month. In anotherembodiment, the invention provides a method of providing a therapeuticformulation to a subject in need in which the therapeutic polypeptide isinterferon, or in which the therapeutic nucleic acid encodes,interferon, which in one embodiment, is interferon alpha, which in oneembodiment, is interferon alpha 2a. In another embodiment, the inventionprovides a method of providing a therapeutic polypeptide which isinterferon, which in one embodiment, is interferon alpha, which in oneembodiment, is interferon alpha 2a to a subject in need.

In one embodiment, interferons are multi-functional cytokines that arecapable of producing pleitrophic effects on cells, such as anti-viral,anti-proliferative and anti-inflammatory effects. Because of thesecellular responses to interferons, interferon-alpha and interferon-betahave been found to be clinically useful in the treatment of viral,proliferative and inflammatory diseases such as multiple sclerosis,hepatitis B, hepatitis C and several forms of cancer. Interferontherapies may also have potential use for the treatment of otherinflammatory diseases, viral diseases and proliferative diseases. Thus,a subject in need of interferons may have one or all of theabove-mentioned diseases or conditions.

There are three major classes of interferons: alpha (α), beta (β), andgamma (γ). Aside from their antiviral and anti-oncogenic properties,interferons activate macrophage and natural killer lymphocyte, andenhance major histocompatibility complex glycoprotein classes I and II.Interferon-α is secreted by leukocytes (B-cells and T-cells).Interferon-β is secreted by fibroblasts, and interferon-γ is secreted byT-cells and natural killer lymphocytes.

In one embodiment, the therapeutic polypeptide is interferon alpha, inanother embodiment, interferon beta, or in another embodiment,interferon gamma. In another embodiment, the therapeutic polypeptide isany subtype of interferon alpha, including but not limited to: 1, 2, 4,5, 6, 7, 8, 10, 13, 14, 16, 17, or 21. In another embodiment, thetherapeutic polypeptide is interferon omega, epsilon, kappa, or ahomolog thereof. In another embodiment, the therapeutic polypeptide isinterferon lambda or a homolog thereof. In another embodiment, thetherapeutic polypeptide is any subtype of interferon lambda includingbut not limited to: Interleukin (IL) 28A, IL28B, or IL29. In anotherembodiment, the therapeutic polypeptide is interferon zeta, nu, tau,delta, or a homolog thereof.

In one embodiment, IFNs bind to a specific cell surface receptorcomplex, which in one embodiment is interferon alpha receptor (IFNAR)comprising IFNAR1 and IFNAR2 chains, in another embodiment is interferongamma receptor (IFNGR) complex, which comprises two IFNGR1 and twoIFNGR2 subunits, in another embodiment is a receptor complex comprisingIL10R2 and IFNLR1. In one embodiment, interferons signal through theJAK-STAT signaling pathway.

In one embodiment, the interferon of the formulations and methods of theinstant invention are interferon alpha. In another embodiment, theinterferon of the formulations and methods of the instant invention areinterferon alpha2b. In one embodiment, IFN-alpha-2b is a recombinant,non-glycosylated 165-amino acid alpha interferon protein comprising thegene for IFN-alpha-2b from human leukocytes. IFN-alpha-2b is a type I,water-soluble interferon with a molecular weight of 19,271 daltons(19.271 kDa). In one embodiment, IFN-alpha-2b has a specific activity ofabout 2.6×108 (260 million) International Units/mg as measured by HPLCassay.

In one embodiment, IFN-alpha-2b is one of the Type I interferons, whichbelong to the larger helical cytokine superfamily, which includes growthhormones, interleukins, several colony-stimulating factors and severalother regulatory molecules. All function as regulators of cellularactivity by interacting with cell-surface receptor complexes, known asIFNAR1 and IFNAR2, and activating various signaling pathways.Interferons produce antiviral and anti-proliferative responses in cells.

In one embodiment, a long-lasting IFN-alpha formulation of the presentinvention may be used for the prevention or treatment of hairy cellleukemia, venereal warts, Kaposi's Sarcoma, chronic non-A, non-Bhepatitis, hepatitis B, or a combination thereof. In another embodiment,a long-lasting IFN-alpha formulation of the present invention may beadministered to a subject that is susceptible to one of theabove-mentioned diseases or conditions or has been or will be exposed toan infectious agent, as described herein. In another embodiment, along-lasting IFN-alpha formulation invention may be used for theprevention or treatment of hepatitis C. According to this aspect and inone embodiment, the formulations of the present invention may beadministered concurrently or alternately with other hepatitis Ctreatments, including inter alia, ribavarin, interferons, pegylatedinterferons or a combination thereof.

In another embodiment, a long-lasting IFN-alpha formulation may be usedor evaluated alone or in conjunction with chemotherapeutic agents in avariety of other cellular proliferation disorders, including chronicmyelogenous leukemia, multiple myeloma, superficial bladder cancer, skincancers (including, inter alia, basal cell carcinoma and malignantmelanoma), renal cell carcinoma, ovarian cancer, low grade lymphocyticand cutaneous T cell lymphoma, and glioma. In another embodiment, along-lasting IFN-alpha formulation may be used for the prevention ortreatment of solid tumors that arise from lung, colorectal and breastcancer, alone or with other chemotherapeutic agents. In anotherembodiment, a long-lasting IFN-alpha formulation may be used for theprevention or treatment of multiple sclerosis. In another embodiment, along-lasting IFN-alpha formulation may be used for the prevention ortreatment of histiocytic diseases, which in one embodiment isErdheim-Chester disease (ECD), which in one embodiment is a potentiallyfatal disorder that attacks the body's connective tissue and in oneembodiment is caused by the overproduction of histiocytes, which in oneembodiment, accumulate in loose connective tissue, causing it to becomethickened and dense. In another embodiment, a long-lasting IFN-alphaformulation may be used for the prevention or treatment of severe ocularBehcet's disease.

In one embodiment, the interferon alpha gene has a nucleic acid sequencethat corresponds to that set forth in Genbank Accession Nos: K01900;M11003; or M71246, or encodes a protein sequence that corresponds tothat set forth in Genbank Accession Nos: AAA52716; AAA52724; orAAA52713. In one embodiment, the interferon beta gene has a nucleic acidsequence that corresponds to that set forth in Genbank Accession Nos:M25460; AL390882; or CH236948, or encodes a protein sequence thatcorresponds to that set forth in Genbank Accession Nos: AAC41702;CAH70160; or EAL24265. In one embodiment, the interferon gamma gene hasa nucleic acid sequence that corresponds to that set forth in GenbankAccession Nos: J00219; AF506749; NM_(—)000619; or X62468, or encodes aprotein sequence that corresponds to that set forth in Genbank AccessionNos: AAB59534; AAM28885; NP_(—)000610; or CAA44325. In anotherembodiment, the interferon alpha gene has a nucleic acid sequence aspresented in SEQ ID No: 9, while in another embodiment, the interferonalpha gene has an amino acid sequence as presented in SEQ ID No: 10. Inanother embodiment, the interferon alpha gene has a nucleic acid that ishomologous to that presented in SEQ ID No: 9, while in anotherembodiment, the interferon alpha gene has an amino acid sequence that ishomologous to that presented in SEQ ID No: 10.

In another embodiment, the present invention provides a method ofdelivering interferon-alpha to a subject in need over a sustained periodcomprising: providing one or more genetically modified micro-organs,said micro-organs comprising a vector comprising a nucleic acid sequenceoperably linked to one or more regulatory sequences; and implanting saidgenetically modified micro-organ in said subject, wherein said nucleicacid sequence encodes interferon-alpha and whereby interferon-alphalevels are increased by more than 5% over basal levels and saidincreased interferon-alpha levels persist for greater than one month.

In one embodiment, the formulations and methods of the present inventionprovide a nucleic acid optimized for increased expression levels,duration, or a combination thereof of a therapeutic polypeptide encodedby said nucleic acid. In another embodiment, the invention provides anucleic acid sequence with greater than 85% homology to SEQ ID No: 1, avector comprising such a nucleic acid sequence, and a cell comprisingsuch as vector.

In another embodiment, the invention provides a nucleic acid sequencewith greater than 85% homology to SEQ ID No: 2, a vector comprising sucha nucleic acid sequence, and a cell comprising such as vector.

The term “homology”, as used herein, when in reference to any nucleicacid sequence indicates a percentage of nucleotides in a candidatesequence that is identical with the nucleotides of a correspondingnative nucleic acid sequence.

In one embodiment, the terms “homology”, “homologue” or “homologous”, inany instance, indicate that the sequence referred to, exhibits, in oneembodiment at least 70% correspondence with the indicated sequence. Inanother embodiment, the nucleic acid sequence exhibits at least 72%correspondence with the indicated sequence. In another embodiment, thenucleic acid sequence exhibits at least 75% correspondence with theindicated sequence. In another embodiment, the nucleic acid sequenceexhibits at least 77% correspondence with the indicated sequence. Inanother embodiment, the nucleic acid sequence exhibits at least 80%correspondence with the indicated sequence. In another embodiment, thenucleic acid sequence exhibits at least 82% correspondence with theindicated sequence. In another embodiment, the nucleic acid sequenceexhibits at least 85% correspondence with the indicated sequence. Inanother embodiment, the nucleic acid sequence exhibits at least 87%correspondence with the indicated sequence. In another embodiment, thenucleic acid sequence exhibits at least 90% correspondence with theindicated sequence. In another embodiment, the nucleic acid sequenceexhibits at least 92% correspondence with the indicated sequence. Inanother embodiment, the nucleic acid sequence exhibits at least 95% ormore correspondence with the indicated sequence. In another embodiment,the nucleic acid sequence exhibits 95%-100% correspondence to theindicated sequence. Similarly, reference to a correspondence to aparticular sequence includes both direct correspondence, as well ashomology to that sequence as herein defined.

Homology may be determined by computer algorithm for sequence alignment,by methods well described in the art. For example, computer algorithmanalysis of nucleic acid sequence homology may include the utilizationof any number of software packages available, such as, for example, theBLAST, DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), GENPEPT andTREMBL packages.

An additional means of determining homology is via determination ofnucleic acid sequence hybridization, methods of which are well describedin the art (See, for example, “Nucleic Acid Hybridization” Hames, B. D.,and Higgins S. J., Eds. (1985); Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, (Volumes 1-3) Cold Spring Harbor Press,N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology,Green Publishing Associates and Wiley Interscience, N.Y). In oneembodiment, methods of hybridization may be carried out under moderateto stringent conditions. Hybridization conditions being, for example,overnight incubation at 42° C. in a solution comprising: 10-20%formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH 7. 6), 5×Denhardt's solution, 10% dextran sulfate, and 20μg/ml denatured, sheared salmon sperm DNA.

In one embodiment, the present invention provides therapeuticformulations comprising micro-organs and methods of use thereof. In oneembodiment, the preparation of therapeutic micro-organs comprises (a)obtaining a plurality of micro-organ explants from a donor subject, eachof the plurality of micro-organ explants comprises a population ofcells, each of the plurality of micro-organ explants maintaining amicroarchitecture of an organ from which it is derived and at the sametime having dimensions selected so as to allow diffusion of adequatenutrients and gases to cells in the micro-organ explants and diffusionof cellular waste out of the micro-organ explants so as to minimizecellular toxicity and concomitant death due to insufficient nutritionand accumulation of the waste in the micro-organ explants; (b)genetically modifying the plurality of micro-organ explants, so as toobtain a plurality of genetically modified micro-organ explants, saidmicro-organs comprising and secreting the proteins differing by the atleast one amino acid; and (c) implanting the plurality of geneticallymodified micro-organ explants within a plurality of recipient subjects.

In one embodiment, the preparation of therapeutic micro-organs isperformed as described in PCT patents WO 03/006669, WO 03/03585, and WO04/0993631, which are incorporated herein by reference in theirentirety.

Methods for the preparation and processing of micro-organs intogenetically modified micro-organs are disclosed in WO2004/099363,incorporated herein by reference in their entirety. Micro-organscomprise tissue dimensions defined such that diffusion of nutrients andgases into every cell in the three dimensional micro-organ, andsufficient diffusion of cellular wastes out of the explant, is assured.Ex vivo maintenance of the micro-organs, which in one embodiment, is inminimal media, can continue for an extended period of time, whereuponcontrolled ex vivo transduction incorporating desired gene candidateswithin cells of the micro-organs using viral or non-viral vectorsoccurs, thus creating genetically modified micro-organs.

In one embodiment, micro-organs are harvested using a drill and coringneedle, as described hereinbelow. In another embodiment, micro-organsare harvested using a harvesting system that utilizes a vacuum to holdthe skin taut and open the slits during insertion of the coring drill.In another embodiment, any tool which may be used to harvest dermaltissue may be used to harvest micro-organs of the appropriate size,including but not limited to those tools and methods described in PCTApplication WO 04/099363.

Incorporation of recombinant nucleic acid within the micro-organs togenerate genetically modified micro-organs or biopumps can beaccomplished through a number of methods well known in the art. Nucleicacid constructs can be utilized to stably or transiently transduce themicro-organ cells. In stable transduction, the nucleic acid molecule isintegrated into the micro-organ cells genome and as such it represents astable and inherited trait. In transient transduction, the nucleic acidmolecule is maintained in the transduced cells as an episome and isexpressed by the cells but it is not integrated into the genome. Such anepisome can lead to transient expression when the transduced cells arerapidly dividing cells due to loss of the episome or to long termexpression wherein the transduced cells are non-dividing cells.

Typically the nucleic acid sequence is subcloned within a particularvector, depending upon the preferred method of introduction of thesequence to within the micro-organs, as described hereinabove. Once thedesired nucleic acid segment is subcloned into a particular vector itthereby becomes a recombinant vector.

In one embodiment, micro-organs are incubated at 32° C. before and aftergenetic modification, while in another embodiment, they are incubated at37° C. In another embodiment, micro-organs are incubated at 33° C., 34°C., 35° C., 36° C., 38° C., 39° C., 40° C., 28° C., 30° C., 31° C., 25°C., 42° C., or 45° C.

In one embodiment, micro-organs are incubated at 10% CO₂ before andafter genetic modification, while in another embodiment, they areincubated at 5% CO₂. In another embodiment, micro-organs are incubatedat 12% CO₂, 15% CO₂, 17% CO₂, or 20% CO₂. In another embodiment,micro-organs are incubated at 2% CO₂, 6% CO₂, 7% CO₂, 8% CO₂, or 9% CO₂

In another embodiment, incubation temperatures, CO₂ concentrations, or acombination thereof may be kept at a single temperature or concentrationbefore, during, and after genetic modification, while in anotherembodiment, incubation temperatures, CO₂ concentrations, or acombination thereof may be adjusted at different points before, during,and after genetic modification of micro-organs.

In another embodiment, micro-organs are incubated at 85-100% humidity,which in one embodiment is 95% humidity, in another embodiment, 90%humidity, and in another embodiment, 98% humidity.

In one embodiment, the levels of therapeutic nucleic acids orpolypeptides may be detected using any method known in the art. Theefficacy of a particular expression vector system and method ofintroducing nucleic acid into a cell can be assessed by standardapproaches routinely used in the art. For example, DNA introduced into acell can be detected by a filter hybridization technique (e.g., Southernblotting) and RNA produced by transcription of introduced DNA can bedetected, for example, by Northern blotting, RNase protection or reversetranscriptase-polymerase chain reaction (RT-PCR). The gene product canbe detected by an appropriate assay, for example by immunologicaldetection of a produced protein, such as with a specific antibody, or bya functional assay to detect a functional activity of the gene product,such as an enzymatic assay. In one embodiment, ELISA, Western blots, orradioimmunoassay may be used to detect proteins. If the gene product ofinterest to be expressed by a cell is not readily assayable, anexpression system can first be optimized using a reporter gene linked tothe regulatory elements and vector to be used. The reporter gene encodesa gene product which is easily detectable and, thus, can be used toevaluate efficacy of the system. Standard reporter genes used in the artinclude genes encoding β-galactosidase, chloramphenicol acetyltransferase, luciferase and human growth hormone.

Thus, in one embodiment, therapeutic polypeptide or nucleic acidexpression levels are measured in vitro, while in another embodiment,therapeutic polypeptide or nucleic acid expression levels are measuredin vivo. In one embodiment, in vitro determination of polypeptide ornucleic acid expression levels, which in one embodiment, is EPO levelsand in another embodiment, IFN-alpha levels, allows a determination ofthe number of micro organs to be implanted in a patient via determiningthe secretion level of a therapeutic agent by a micro-organ in vitro;estimating a relationship between in vitro production and secretionslevels and in vivo serum levels of the therapeutic agent; anddetermining an amount of the therapeutic formulation to be implanted,based on the determined secretion level and the estimated relationship.

In another preferred embodiment of this invention, polynucleotide(s) canalso include trans-, or cis-acting enhancer or suppresser elements whichregulate either the transcription or translation of endogenous genesexpressed within the cells of the micro-organs, or additionalrecombinant genes introduced into the micro-organs. Numerous examples ofsuitable translational or transcriptional regulatory elements, which canbe utilized in mammalian cells, are known in the art.

For example, transcriptional regulatory elements comprise cis- ortrans-acting elements, which are necessary for activation oftranscription from specific promoters [(Carey et al., (1989), J. Mol.Biol. 209:423-432; Cress et al., (1991), Science 251:87-90; and Sadowskiet al., (1988), Nature 335:5631-564)].

Translational activators are exemplified by the cauliflower mosaic virustranslational activator (TAV) [see for example, Futterer and Hohn,(1991), EMBO J. 10:3887-3896]. In this system a bi-cistronic mRNA isproduced. That is, two coding regions are transcribed in the same mRNAfrom the same promoter. In the absence of TAV, only the first cistron istranslated by the ribosomes, however, in cells expressing TAV, bothcistrons are translated.

The polynucleotide sequence of cis-acting regulatory elements can beintroduced into cells of micro-organs via commonly practiced geneknock-in techniques. For a review of gene knock-in/out methodology see,for example, U.S. Pat. Nos. 5,487,992, 5,464,764, 5,387,742, 5,360,735,5,347,075, 5,298,422, 5,288,846, 5,221,778, 5,175,385, 5,175,384,5,175,383, 4,736,866 as well as Burke and Olson, Methods in Enzymology,194:251-270, 1991; Capecchi, Science 244:1288-1292, 1989; Davies et al.,Nucleic Acids Research, 20 (11) 2693-2698, 1992; Dickinson et al., HumanMolecular Genetics, 2(8):1299-1302, 1993; Duff and Lincoln, “Insertionof a pathogenic mutation into a yeast artificial chromosome containingthe human APP gene and expression in ES cells”, Research Advances inAlzheimer's Disease and Related Disorders, 1995; Huxley et al.,Genomics, 9:742-750 1991; Jakobovits et al., Nature, 362:255-261 1993;Lamb et al., Nature Genetics, 5: 22-29, 1993; Pearson and Choi, Proc.Natl. Acad. Sci. USA, 1993, 90:10578-82; Rothstein, Methods inEnzymology, 194:281-301, 1991; Schedl et al., Nature, 362: 258-261,1993; Strauss et al., Science, 259:1904-1907, 1993, WO 94/23049, WO93/14200, WO 94/06908 and WO 94/28123 also provide information.

Down-regulation of endogenous sequences may also be desired, in order toassess production of the recombinant product exclusively. Toward thisend, antisense RNA may be employed as a means of endogenous sequenceinactivation. Exogenous polynucleotide(s) encoding sequencescomplementary to the endogenous mRNA sequences are transcribed withinthe cells of the micro-organ. Down regulation can also be effected viagene knock-out techniques, practices well known in the art (“MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988)).

Over expression of the recombinant product may be desired as well.Overexpression may be accomplished by providing a high copy number ofone or more coding sequences in the respective vectors. These exogenouspolynucleotide sequences can be placed under transcriptional control ofa suitable promoter of a mammalian expression vectors to regulate theirexpression. In another embodiment, multiple copies of the same gene orof several related genes may be used as a means to increase polypeptideor nucleic acid expression. In one embodiment, expression is stabilizedby DNA elements, which in one embodiment are matrix-associating regions(MARs) or scaffold-associating regions (SARs).

In one embodiment, an adenoviral vector is the vector of thecompositions and for use in the methods of the present invention. In anembodiment in which an adenoviral vector is used as a vector, thehelper-dependent adenovirus system may be used in one embodiment, toprepare therapeutic polypeptide or nucleic acid-expressinghelper-dependent adenovirus vector for transforming micro-organs. In oneembodiment, such a helper-dependent adenovirus system comprises ahelper-dependent adenovirus, a helper virus, and a producer cell line isused in the preparation of the formulation of the present invention isas described in Palmer and Ng, 2003 Mol Ther 8:846 and in Palmer and Ng,2004 Mol Ther 10:792, which are hereby incorporated herein by referencein their entirety.

In one embodiment, a helper cell line, designated 293, which wastransformed from human embryonic kidney cells by Ad5 DNA fragments andconstitutively expresses E1 proteins is used to generate and propagatereplication deficient adenoviral vectors. In another embodiment, helpercell lines may be derived from human muscle cells, hematopoietic cellsor other human embryonic mesenchymal or epithelial cells. Alternatively,the helper cells may be derived from the cells of other mammalianspecies that are permissive for human adenovirus. Such cells include,e.g., Vero cells or other monkey embryonic mesenchymal or epithelialcells.

In one embodiment, micro-organs are maintained ex vivo for a period oftime, which may range from several hours to several months. In oneembodiment, they are maintained for several days, and in anotherembodiment, for several weeks prior to implantation. Without beinglimited by theory, in one embodiment, said incubation allows cells toprocess and break down viral proteins, which in one embodiment are viralcapsids, present as a result of viral vector transduction. In oneembodiment, such a turnover of capsid proteins occurs within 2-3 days,so that, in one embodiment, little if any viral capsid proteins remainby the 10^(th) day ex vivo. In one embodiment, the breaking down ofviral capsids further reduces the immunogenicity of the formulations ofthe instant invention and increases the expression levels and expressionduration of the gene or genes of interest. In another embodiment, saidincubation allows the early HD-Ad vector-induced innate immune responsesto occur in vitro, which in one embodiment, will not persist beyond 24hours in the absence of Adeno gene transcription. In another embodiment,the later adaptive responses that normally follow the administration oftranscription-competent first-generation-Ad vectors, which arepredominantly characterized in one embodiment, by lymphocyteinfiltration and in another embodiment by induction of Ad-specificCTL's, are not be elicited by HD-Ad vectors.

In one embodiment, the ex vivo micro-organ is exposed to viral vector ata dosage of 1.6-3×10⁹ infectious particles (ip)/ml, 3-4×10¹² viralparticles/ml, or 2×10¹¹ viral particles/ml. In another embodiment, exvivo micro-organs are exposed to viral vector at a dosage of 1×10³ to1×10¹² viral particles/ml, in another embodiment from 1×10³ to 1×10⁹,and in another embodiment, from 1×10⁶ to 1×10⁹ and in anotherembodiment, 1×10⁶ to 1×10¹² viral particles/ml. In one embodiment, thedosage of viral particles/g body weight of subject that are administeredto a subject within a micro-organ is less than 1×10³, and in anotherembodiment, less than 1×10², and in another embodiment, less than 1×10¹viral particles/g body weight of subject.

In one embodiment, growth factors are used to increase the number ofcells in the micro-organs.

In one embodiment, in vitro expression can be assessed prior toimplantation, enabling the possibility for in vitro to in vivocorrelation studies of expressed recombinant proteins.

In some embodiments of the invention, the amounts of tissue sampleincluding a genetically modified cell(s) to be implanted are determinedfrom one or more of: corresponding amounts of the therapeutic agent ofinterest routinely administered to such subjects based on regulatoryguidelines, specific clinical protocols or population statistics forsimilar subjects, corresponding amounts of the therapeutic agent such asprotein of interest specifically to that same subject in the case thathe/she has received it via injections or other routes previously,subject data such as weight, age, physical condition, clinical status,pharmacokinetic data from previous tissue sample which includes agenetically modified cell administration to other similar subjects,response to previous tissue sample which includes a genetically modifiedcell administration to that subject, or a combination thereof. Thus, inone embodiment, the level of expression of gene products by one or moremicro-organs is determined in vitro, a relationship between in vitro andin vivo therapeutic polypeptide or nucleic acid expression levels isdetermined or estimated, and the number of micro-organs to be implantedin a particular patient is determined based on the calculated orestimated relationship. The dosage of the therapeutic agent may beadjusted as described previously (WO2004/099363).

In one embodiment, a micro-organ or a genetically modified micro-organmay be maintained in vitro for a proscribed period of time until theyare needed for implantation into a host. In one embodiment, amicro-organ or a genetically modified micro-organ may be maintained orstored in culture for between 1-7 days, between 1-8 weeks, or for 1-4months. In another embodiment, the therapeutic agent, left in thesupernatant medium surrounding the tissue sample, can be isolated andinjected or applied to the same or a different subject.

Alternatively or additionally, a genetically modified micro-organ can becryogenically preserved by methods known in the art, for example,without limitation, gradual freezing (0° C., −20° C., −80° C., −196° C.)in DMEM containing 10% DMSO, immediately after being formed from thetissue sample or after genetic alteration.

In one embodiment, the formulation of the instant invention may beimplanted in an organ or system that is affected by a disease ordisorder to be treated or prevented by a method or route which resultsin localization of the micro-organ at a desired site. In anotherembodiment, the location of the implanted formulation may be distal froman organ or system that is affected by a disease or disorder. Thus,while in one embodiment, the recombinant protein is released locally, inanother embodiment, the recombinant protein diffuses to the lymphaticsystem, which in one embodiment, may ultimately lead to systemicdistribution of the recombinant protein. Thus, the present inventionprovides for the use of therapeutic formulations in variousconcentrations to treat a disease or disorder manifesting in any part ofthe subject in need.

According to this aspect and in one embodiment, formulations of theinstant invention may be implanted intratumorally. In anotherembodiment, formulations may be implanted at a site distal from thetumor, which in one embodiment is associated with metastasis of aparticular type of tumor. In another embodiment, formulations of theinstant invention may be implanted into the kidney of a subject, whichin one embodiment is a subcapsular implantation. In another embodiment,formulations of the instant invention are implanted laparascopically.

In one embodiment, the formulations of the invention may be implanted asingle time for acute treatment of temporary conditions, or may beimplanted more than one time, especially in the case of progressive,recurrent, or degenerative disease. In one embodiment, one or moreformulations of the invention may be administered simultaneously, or inanother embodiment, they may be administered in a staggered fashion. Inone embodiment, the staggered fashion may be dictated by the stage orphase of the disease.

In one embodiment, the micro-organ is implanted at a desired location inthe subject in such a way that at least a portion of the cells of themicro-organ remain viable. In one embodiment of this invention, at leastabout 5%, in another embodiment of this invention, at least about 10%,in another embodiment of this invention, at least about 20%, in anotherembodiment of this invention, at least about 30%, in another embodimentof this invention, at least about 40%, and in another embodiment of thisinvention, at least about 50% or more of the cells remain viable afteradministration to a subject. The period of viability of the cells afteradministration to a subject can be as short as a few hours, e.g.,twenty-four hours, to a few days, to as long as a few weeks to months oryears.

Micro-organ implantation within a recipient subject provides for asustained dosage of the recombinant product. The micro-organs may beprepared, prior to implantation, for efficient incorporation within thehost facilitating, for example, formation of blood vessels within theimplanted tissue. Recombinant products may therefore be deliveredimmediately to peripheral recipient circulation, following production.Alternatively, micro-organs may be prepared, prior to implantation, toprevent cell adherence and efficient incorporation within the host.Examples of methods that prevent blood vessel formation includeencasement of the micro-organs within commercially availablecell-impermeant diameter restricted biological mesh bags made of silk ornylon, or others such as, for example GORE-TEX bags (Terrill P J,Kedwards S M, and Lawrence J C. (1991) The use of GORE-TEX bags for handburns. Burns 17(2): 161-5), or other porous membranes that are coatedwith a material that prevents cellular adhesion, for example Teflon.

Gene products produced by micro-organs can then be delivered via, forexample, polymeric devices designed for the controlled deliverycompounds, e.g., drugs, including proteinaceous biopharmaceuticals. Avariety of biocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a gene product of the micro-organsin context of the invention at a particular target site. The generationof such implants is generally known in the art (see, for example,Concise Encyclopedia of Medical & Dental Materials, ed. By DavidWilliams (MIT Press: Cambridge, Mass., 1990); Sabel et al. U.S. Pat. No.4,883,666; Aebischer et al. U.S. Pat. No. 4,892,538; Aebischer et al.U.S. Pat. No. 5,106,627; Lim U.S. Pat. No. 4,391,909; and Sefton U.S.Pat. No. 4,353,888).

Implantation of genetically modified micro-organs according to thepresent invention can be effected via standard surgical techniques orvia injecting micro-organ preparations into the intended tissue regionsof the mammal utilizing specially adapted syringes employing a needle ofa gauge suitable for the administration of micro-organs. In anotherembodiment, a catheter is employed for implanted micro-organs. In oneembodiment, any of the implantation methods described in PCT PublicationWO2 04/099363 may be used and is considered an embodiment of thisinvention.

In one embodiment, micro-organs are implanted subcutaneously,intradermally, intramuscularly, intraperitoneally or intragastrically.In one embodiment, the term implanted excludes being grafted as asplit-thickness or full-thickness skin graft. In one embodiment of thepresent invention, the donor micro-organs utilized for implantation arepreferably prepared from an organ tissue of the recipient mammal (i.e.autologous), or a syngeneic mammal, although allogeneic and xenogeneictissue can also be utilized for the preparation of the micro-organsproviding measures are taken prior to, or during implantation, so as toavoid graft rejection and/or graft versus host disease (GVHD). As usedherein, GVHD refers to graft versus host disease, a consequence oftissue transplantation (the graft) caused by the transplant immuneresponse against the recipient host. More specifically,graft-versus-host disease is caused by donor T-lymphocytes (T cells),recognizing the recipient as being foreign and attacking cells of therecipient. Numerous methods for preventing or alleviating graftrejection or GVHD are known in the art and may be used in the methods ofthis invention. In one embodiment, to facilitate transplantation of thecell populations within a tissue which may be subject to immunologicalattack by the host, e.g., where xenogenic grafting is used, such asswine-human transplantations, the micro-organ may be inserted into orencapsulated by biocompatible immuno-protected material such asrechargeable, non-biodegradable or biodegradable devices and thentransplanted into the recipient subject.

In another embodiment, the donor micro-organs utilized for implantationare preferably prepared from a donor who is human leukocyte antigen(HLA)-matched with the recipient, where in one embodiment, HLA is themajor histocompatibility complex in humans. In one embodiment, donor andrecipient are matched for class I major histocompatibility complex (MHC)genes, class II MHC genes, or a combination thereof. In one embodiment,class I MHC genes comprise HLA-A, HLA-B, and HLA-C, wherein in oneembodiment, a mismatch of class I MHC genes increases the risk of graftrejection, and in one embodiment, class II MHC genes comprise HLA-DPA1,HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, wherein in oneembodiment, a mismatch of class II MHC genes increases the risk of GVHD.In another embodiment, donor and recipient are matched for HLA-DM andHLA-DO genes.

In one embodiment, viral turnover or elimination from cells ex vivo isenhanced via techniques know in the art, such as physical methods, whichin one embodiment is heating, use of antiviral agents, agents whichstimulate viral turnovers by cells, etc.

In one embodiment, while the long-lasting formulations of the presentinvention increase the level and duration of nucleic acid or polypeptideexpression, the levels of nucleic acid or polypeptide expression do notremain elevated indefinitely. Without being limited by theory, in oneembodiment, levels of nucleic acid or polypeptide expressed by thelong-lasting formulations of the present invention may decrease as afunction of time as a result of the death of differentiated dermalfibroblasts expressing the recombinant nucleic acid or polypeptide.

EXAMPLES Experimental Materials and Methods Materials and Equipment List

Production medium was used to grow micro-organs and comprises DMEM-HEPESMedium (High glucose 4,500 mg/L and 25 mM HEPES; Hi-Clone Cat#SH3A1448.02) comprising 1% glutamine and supplemented with 50 μg/mlGentamycin (RAFA labs, for injection) and 0.1% Amphotericin B (BMS,Fungizone I.V.) (final concentration in the media 2.5 μg/ml AmphotericinB). In some experiments, 10% serum substitute supplement (SSS, IrvineScientific, Cat # 99193), 10% autologous human serum, or 10% Fetalbovine serum (FBS or FCS) was added to the production medium.

Harvesting of Dermal Micro-Organs—Concentric Needle Method

Human dermal micro-organs were harvested from an area of skin from aregion of the donor's lower abdomen. To prevent the harvest of theepidermis, a shallow slit (1-2 mm deep) passing through the stratumcornea into the dermis was cut along a straight line at one side of theskin region from which the micro-organs were to be harvested, and asimilar slit was cut 30 mm away from and parallel to the first slit. Thedistance between the slits determined the micro-organ length and wasconsistent throughout the experiments.

A thin gauge (typically 22GA) hypodermic needle attached to a 1 mlsyringe filled with sterile saline was inserted into the exposed dermisat the first slit and slid along the dermis of the harvesting sitetowards the opposite slit, with the needles angled as necessary so thatit exited through the dermis at the opposite slit.

Next, the outer skin along the length of the guiding needle is pinchedwith a surgical clamp. The needle embedded in the dermis is liftedslightly to raise the area of skin surrounding it and sometimes a hookshaped device beneath the inserted hypodermic needle's point is used toassist in lifting the skin before it's pinched. The tip of the guideneedle protruding from its point of exit, is inserted into the sharpleading end of a coring needle (1-3 mm in diameter, Point Technologies,CO USA), which is held by a commercially available drill (such asAesculap Micro Speed GD 650, GD 657). A small amount of sterile salineis injected from the syringe into the coring needle. The drill isactivated to rotate the coring needle at high speed (typically 3000-7000RPM) and while rotating, the drill and coring needle are manually urgedforward along the axis of the guide needle to cut a 30-40 mm longcylindrical dermal core (dermal micro-organ) having an outer diameterapproximately that of the inner diameter of the coring needle. Thedermal micro-organ usually remains attached to the guide needle, whichis withdrawn from within the coring needle and placed in Productionmedia (as described hereinabove), and the coring needle is removed fromthe skin.

Using tweezers, each micro-organ is transferred to a labeled single wellin a 24 well plate containing 1000 μl Production Medium. To remove thedebris, two additional media changes of 1000 μl are performed for eachmicro-organ. The plates containing the micro-organs in 1000 μlproduction media are then transferred to an incubator that had beenequilibrated to 32° C., 10% CO₂, and ˜95% humidity for a 24 hr recoveryperiod.

Virus Transduction

Each micro-organ was transferred for transduction into a well of a48-well plate, which have smaller wells requiring smaller total fluidvolume, to conserve virus. The medium was carefully removed from eachwell without disturbing the micro-organ inside. During the preclinicalexperiments, three different vectors were tested: 1.6-3×10⁹ infectiousparticles (ip)/ml of first generation adenovirus (Molecular Medicine),approximately 3-4×10¹² viral particles/ml helper-dependent adenovirus(Baylor), or approximately 2×10¹¹ viral particles/ml adeno-associatedvirus (University of Pennsylvania), each comprising recombinant humanEPO gene, optimized recombinant human EPO gene, or optimized IFN-alphagene, were each diluted 1:10, 1:25, 1:50, 1:100, 1:500, or 1:1000 inDMEM-HEPES (Gibco Cat# 42430-025) with or without FCS. Each well of the48-well plates was filled with 100 μL of one of the diluted titers of avirus. The plate was placed in a CO₂ incubator and transduction wasassisted by agitation on a digital microtiter shaker at 300 rpm for aperiod of 2 hours and an additional 16-22 hour incubation withoutshaking.

The transduced micro-organs were transferred to a 24-well plate aftertransduction and then washed three times with 1 mL production media(without FCS) to remove the non-transduced viral particles. Afterwashing, the biopumps were maintained in 1 mL production media in astandard high humidity CO₂ incubator at 95% humidity, 10% CO₂, and 32°C.

Seventy-two hours after the removal of the viral vector, the productionmedium was replaced with fresh medium, and aliquots of the spent mediumwere assayed for secreted recombinant protein levels.

Ex Vivo Micro-Organ Maintenance

Every 3-4 days, used production media was collected, and the level ofthe secreted recombinant protein and glucose level were assessed alongwith the viability of the biopumps. Fresh Production media was added tothe 24-well plate.

Secreted Protein Measurements

Human EPO (hEPO) and IFNαconcentration and secretion levels were assayedusing an enzyme-linked immunosorbent assay (ELISA) kit (Quantikine humanerythropoietin; R&D Systems; Human interferon alpha ELISA kit, PBLBiomedical Laboratories), according to the manufacturer's instructions.

Glucose Measurements

Tissue glucose consumption was evaluated using Sigma-Aldrich CorporationGAGO20 Glucose (GO) Assay Kit, according to manufacturer's instructions.

Hematocrit Measurements

Tissue glucose consumption was evaluated using Sigma-Aldrich CorporationGAGO20 Glucose (GO) Assay Kit, according to manufacturer's instructions.

Hematocrit levels were assayed using centrifugation using the referencemethod recommended by The National Committee for Clinical LaboratoryStandards (NCCLS), as is known in the art. To determine the hematocrit,whole blood in a tube was centrifuged at 10-15,000×g for 5 minutes topellet the red cells (called packed erythrocytes), and the ratio of thecolumn of packed erythrocytes to the total length of the sample in thecapillary tube was measured with a graphic reading device within 10minutes of centrifugation.

Micro-Organ Implantation

In some experiments, genetically modified or control micro-organs wereimplanted subcutaneously in Severe Combined ImmunoDeficiency (SCID) miceafter assaying tissue glucose consumption to ascertain that micro-organswere viable. Male and female SCID mice weighing around 25 grams wereanaesthetized with 140 μl of diluted Ketaset (ketamine HCl) (400 μlKetaset and 600 μl saline) and control or EPO-expressing micro-organswere implanted subcutaneously ten days following micro-organtransduction.

Example 1 EPO and IFNα, Levels Produced In Vitro by GMMOS

Micro-organs were prepared as described above and transduced with ahelper-dependent adenoviral vector expressing an optimzed IFNα genelinked to a CAG promoter, as described above. GMMOs were then maintainedin culture, and the levels of IFNα produced were evaluated by ELISA.Optimized IFNα-expressing micro-organs produced greater than 1000 ng/dayof IFNα in vitro (FIG. 1) for at least 40 days post-harvesting, andrecombinant hEPO-expressing micro-organs produced greater than 1000ng/day of hEPO in vitro (FIGS. 2A-B) for at least 142 dayspost-harvesting.

GMMOs comprising a gutless adenovirus vector encoding optimized hEPOmaintained higher percentages of peak expression for more than 200 dayscompared to micro-organs comprising an adenovirus-5 vector encoding hEPO(FIG. 3). Micro-organs comprising a gutless adenovirus vector encodingoptimized hEPO also maintained a higher percentage of peak expressionfor a longer period of time than micro-organs comprising a gutlessadenovirus vector encoding non-optimized hEPO (FIG. 4). Finally,micro-organs comprising a gutless adenovirus vector encoding hEPOdownstream of a CAG promoter showed higher levels of hEPO expression,which grew more pronounced as a function of post-transduction day,compared to micro-organs comprising a gutless adenovirus vector encodinghEPO downstream of a CMV promoter (FIG. 5).

Example 2 EPO Levels Produced by Human EPO-Expressing GMMOS MaintainedIn Vitro and in Serum of Implanted SCID Mice

EPO-expressing micro-organs were prepared as described above. After atotal of nine days in culture, the amount of EPO produced permicro-organ was measured, and this value was used to determine that eachmouse was implanted with micro-organs expressing equivalent levels ofEPO. On the tenth day, two micro-organs were implanted subcutaneouslyinto each SCID mouse and on the first measurement taken after ten days,levels of hEPO measured in the serum of the SCID mice were significantlyabove baseline levels. The levels remained high at least 216 dayspost-implantation and significantly raised hematocrit levels in SCIDmice for at least 157 days (FIG. 6A). Non-implanted EPO-expressingmicro-organs produced from the same donor at the same time as theimplanted EPO-expressing micro-organs but maintained in vitrocontinuously maintained high levels of EPO production (FIG. 6B).Micro-organs transduced with vectors comprising optimized hEPO geneproduced higher levels of EPO than those transduced with recombinanthEPO gene both in vivo (FIG. 6A) and in vitro (FIG. 6B). Control SCIDmice implanted with non-EPO-producing micro-organs showed no increase ofserum EPO levels and no significant changes in hematocrit levels aftermicro-organ implantation compared to pre-implantation (FIG. 6A).Micro-organs comprising EPO-expressing adenovirus-5, which was used as apositive control, was used at a titer of 1:10 compared to a titer of1:100 for micro-organs comprising EPO-expressing optimized ornon-optimized gutless adenovirus.

Example 3 EPO Levels Produced in Genetically ModifiedMicro-Organ-Implanted Human in Clinical Trials

Clinical trials are performed as described previously (Lippin et al.,2005, Blood 106(7):2280-6, incorporated herein by reference in itsentirety) except that the micro-organ will be harvested and geneticallymodified as described hereinabove.

Phase I clinical trials are performed in Israel in which pre-dialysisanemic patients with chronic kidney disease are implanted withautologous hEPO-GMMOs of the sustained type of the present invention. Asingle implantation treatment with GMMO-hEPO is expected to provide 4-6months of effective EPO therapy. Approval for the Phase I/II GMMO hEPOtrial is approved by Israel's Ministry of Health and is conducted at theHadassah Medical facility. All steps regarding the required regulatoryand clinical standards are coordinated with the FDA, in order tofacilitate US based clinical trials.

In preparation for the planned clinical trial, the required preclinicaltoxicological studies in SCID mice are performed. These studies areperformed as was described previously (Brill-Almon et al. MolecularTherapy 12(2), 274-282) with the additional timepoints longer than 20days. The HD-Ad-hEPO vector for the clinical trial is prepared in an FDAGMP compliant facility to be compliant with the FDA guidelines (GMP) asrequired for its use in patients. The GMMOs are implanted for four tosix months, and then removed or ablated at the termination of the trial,or extended if so requested by the PI with the approval of the ethicscommittee.

As shown in Table 1, the toxicology study comprises three groups of SCIDmice, with an equal numbers of male and female subjects. Due to the highmortality of SCID mice, a large number of animals are included in eachgroup.

TABLE 1 Experimental Design Total # # sacrificed # sacrificed #sacrificed GMMO Dose of mice @ 8 wks @ 16 wks @ 24wks 100-150 IU 30 5M,5F 5M, 5F 5M, 5F Epo/day (17M, 17F) 300-450 IU 30 5M, 5F 5M, 5F 5M, 5FEpo/day (17M, 17F) Control-non 30 5M, 5F 5M, 5F 5M, 5F transduced (17M,17F)

In the first group, each animal receives at most a single dermal 30 mmhEPO GMMO or more likely a portion of a GMMO that secretes in the rangeof 100-150 IU EPO/day. Since each mouse weighs approximately 25 grams,this dose equals 4,000-6000 IU/day per kg mouse (25 fold or greater thanthe highest expected dose proposed to implant in human patients). Thesize of the implanted tissue generally corresponds to at least ¼ of awhole GMMO due to the small size of the GMMO and its impact on itshandling.

In the second group, each animal receives at most a single dermal 30 mmhEPO GMMO or more likely a portion of a GMMO that will secrete in therange of 300-450 IU EPO/day. Since a mouse weighs approximately 25grams, this dose equals 12,000-18,000 IU/day per kg mouse (80-120 foldor greater than the highest expected dose proposed to implant in humanpatients).

In the third group, a control group, each animal receives one third (10mm) of a 30 mm dermal non-transduced GMMO.

Dosing rationale: GMMO-hEPO dosing before implantation is controlled byadjusting the numbers or the size of the GMMOs after measuring theactual daily amount of protein produced by the GMMO in vitro. In theexperimental protocol outlined above, the micro-organs are transducedwith a viral titer that is similar to the one which we use in theclinical trial, thereby exposing the cells in the GMMO to a similarmultiplicity of infection. The typical levels of secreted hEPO producedby these GMMOs are in the range of 300-1000 IU/Biopump per day.Therefore, the lowest amount of EPO expected to be secreted from ⅓ BPcorresponding to 1.0 cm and ⅔ BPs corresponding to 2.0 cm isapproximately 100 and 200 IU, respectively. The dose may be lowered inthe mice by implanting fragments shorter than 0.5 cm.

Based on the results of our previous clinical trial, approximately1500-3500 IU/70 kg patient (or 1-3 GMMOs) are expected to be adequate tocause sustained production of reticulocytes and the resulting elevationof hematocrit. Thus, 100 IU of EPO secreted from a single GMMO or afragment of a GMMO implanted in a 25 gr mouse, is at least 25 fold orgreater the highest expected dose that we propose to implant in humanpatients. Thus, using at least ¼ of a GMMO in this study provides asufficient safety margin to test the toxicological effects of GMMO-EPOin the mice and support the clinical dose.

As we've demonstrated, hEPO secretion levels from multiple humanabdominal skin sample GMMOs were approximately 300->1000 IU/day. Whilesecretion levels from GMMOs from the same skin samples were similar, thevariability between different skin samples was higher. As we havedemonstrated the dosing variability is addressed before the GMMOs areimplanted in the mice by measuring the secretion levels in vitro beforeimplantation. The entire study, except histology, will be done underGLP. Histology slides will be reviewed blind by a board certifiedpathologist. Tests to be performed include:

Clinical signs: Daily

Body weight: Every other day

Organ weights: heart, liver, kidney, spleen, brain, thymus (if it can befound) will be determined at terminal sacrifice

Clinical chemistry: At terminal sacrifice

Full hematology profile: At terminal sacrifice. To include serum hEPOlevels and hematocrit.

Clinical pathology: At terminal sacrifice

Histology including: implantation site, liver, kidney, lymph nodes,heart, spleen, bone marrow, and any lesions found at necropsy and bonemarrow will be performed at terminal sacrifice

All other organs will be preserved for future analysis

In contrast to other methods involving transient transduction of cells,or cells that turn over rapidly, the long-lasting EPO formulation of theinstant invention comprises cells that are no longer replicating.Therefore, the EPO formulation produces a stable protein from a stableconstruct and is expected to continue producing the protein alreadycharacterized.

1. A long-lasting therapeutic formulation for implanting into animmunocompetent human subject comprising a genetically modifiedmicro-organ, wherein said micro-organ is transduced in vitro with ahelper-dependent adenoviral vector comprising a nucleic acid sequenceencoding an interferon operably linked to one or more regulatorysequences, wherein following said transduction the genetically modifiedmicro-organ is maintained ex vivo prior to implantation in order toreduce the immunogenicity of said formulation and wherein implantationof said long-lasting formulation in said immunocompetent human subjectprovides a beneficial effect selected from the group consisting of: a.an increase in expression levels of said interferon in the serumcompared with pre-implantation basal levels, said increase persistingfor greater than one month; and b. an alleviation of a symptom of adisease or disorder in said subject, wherein said alleviation persistsfor greater than one month.
 2. The formulation of claim 1, wherein saidregulatory sequence comprises a CAG promoter.
 3. The formulation ofclaim 1, wherein said regulatory sequence comprises a CMV promoter. 4.The formulation of claim 1, wherein said regulatory sequence comprises aSV40 polyadenylation sequence.
 5. The formulation of claim 1, whereinsaid nucleic acid sequence encoding said interferon is optimized forincreased expression levels, increased duration of expression, or acombination thereof.
 6. The formulation of claim 5, wherein saidoptimized nucleic acid sequence is greater than 85% homologous to SEQ IDNo:
 2. 7. The formulation of claim 1, wherein said genetically modifiedmicro-organ is a genetically modified dermal micro-organ.
 8. Theformulation of claim 1, wherein said interferon is interferon alpha. 9.The formulation of claim 8, wherein said interferon alpha is interferonalpha 2a.
 10. The formulation of claim 8, wherein said interferon alphais interferon alpha 2b.
 11. The formulation of claim 1, wherein saidinterferon is interferon beta.
 12. The formulation of claim 1, whereinsaid interferon is interferon gamma.
 13. A method of providing aninterferon to a subject in need over a sustained period comprising thesteps of: a. providing at least one long-lasting formulation of claim 1comprising a genetically modified micro-organ, wherein said micro-organis transduced in vitro with a helper-dependent adenoviral vectorcomprising a nucleic acid sequence encoding an interferon operablylinked to one or more regulatory sequences, wherein following saidtransduction the genetically modified micro-organ is maintained ex vivoprior to implantation in order to reduce the immunogenicity of saidformulation; and b. implanting said genetically modified micro-organ insaid subject, wherein said implanting provides a beneficial effectselected from the group consisting of: i. an increase in expressionlevels of said interferon in the serum compared with pre-implantationbasal levels, said increase persisting for greater than one month; andii. an alleviation of a symptom of a disease or disorder in saidsubject, wherein said alleviation persists for greater than one month.14. The method of claim 13, further comprising the step of determiningsaid interferon expression levels in vitro prior to step (b).
 15. Themethod of claim 13, wherein said nucleic acid sequence encoding saidinterferon is optimized for increased expression levels, increasedduration of expression, or a combination thereof.
 16. The method ofclaim 15, wherein said optimized nucleic acid sequence is greater than85% homologous to SEQ ID No:
 2. 17. The method of claim 13, wherein saidgenetically modified micro-organ is a genetically modified dermalmicro-organ.
 18. The method of claim 13, wherein said interferon isinterferon alpha.
 19. The method of claim 18, wherein said interferonalpha is interferon alpha 2a or interferon alpha 2b.
 20. The method ofclaim 13, wherein said interferon is interferon beta or interferongamma.